Regulated Control of the Assembly and Diversity of LPS by ... · Ugd BasS/R PmrR LpxT EptC Rcs RprA...

Review ArticleRegulated Control of the Assembly and Diversity ofLPS by Noncoding sRNAs

Gracjana Klein and Satish Raina

Unit of Bacterial Genetics Gdansk University of Technology Narutowicza 1112 80-233 Gdansk Poland

Correspondence should be addressed to Satish Raina satishrainapggdapl

Received 10 August 2015 Revised 7 October 2015 Accepted 13 October 2015

Academic Editor Claudio Valverde

Copyright copy 2015 G Klein and S Raina This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

The outer membrane (OM) of Gram-negative bacteria is asymmetric due to the presence of lipopolysaccharide (LPS) facing theouter leaflet of the OM and phospholipids facing the periplasmic side LPS is essential for bacterial viability since it providesa permeability barrier and is a major virulence determinant in pathogenic bacteria In Escherichia coli several steps of LPSbiosynthesis and assembly are regulated by the RpoE sigma factor and stress responsive two-component systems as well as dedicatedsmall RNAs LPS composition is highly heterogeneous and dynamically altered upon stress and other challenges in the environmentbecause of the transcriptional activation of RpoE regulonmembers and posttranslational control by RpoE-regulatedHfq-dependentRybB andMicA sRNAsThe PhoPQ two-component system further regulates Kdo

2-lipid Amodification viaMgrR sRNA Some of

these structural alterations are critical for antibiotic resistance OM integrity virulence survival in host and adaptation to specificenvironmental niches The heterogeneity arises following the incorporation of nonstoichiometric modifications in the lipid A partand alterations in the composition of inner and outer core of LPS The biosynthesis of LPS and phospholipids is tightly coupledThis requires the availability of metabolic precursors whose accumulation is controlled by sRNAs like SlrA GlmZ and GlmY

1 Introduction

Extensive studies in the last decade have established beyonddoubt that in addition to known roles for DNA-bindingtranscriptional regulators noncoding regulatory RNAs playa pivotal role in several key aspects of bacterial physiologyAs most of such RNAs are small in size and majority of themare noncoding RNAs they are commonly referred as smallnoncoding RNAs (sRNAs) They contribute in the regulationof important processes including nutrient uptake transportof specific substrates maintenance of metabolic fluxes ironhomeostasis carbon metabolism stress responses biofilmformation host-cell contact and homeostatic control ofkey components of the cell envelope (reviewed in [1ndash3])Regulatory RNAs can act by different mechanisms modu-lating gene expression either positively or negatively Oneclass of regulatory RNAs comprises riboswitches or RNAthermometers which are part of the same mRNA that theyregulate [4ndash6] Their leader sequence in the 51015840 UTR of

mRNA can adopt different conformations in response toeither ligand binding or changes in nutritional status orspecific stresses such as temperature Interaction with ligandor structural changes upon stress allow for a direct andimmediate response Another very well characterized groupof regulatory RNAs acts by base-pairingwith targetmRNA(s)and modulates either their stability or their translationDepending on their location with respect to their targetthese molecules are designated to act as cis- or trans-actingRNAs [1ndash3] Majority of them were initially identified tobe located in the intergenic regions [7] However recentlyregulatory RNAs have also been identified in the 31015840 UTRof mRNAs [8 9] Many of these sRNAs require the RNAchaperone Hfq for function Hfq acts by facilitating base-pairing between sRNAs and target mRNAs [1ndash3] Hfq isalso required for stabilization of sRNAs by protecting themfrom RNA degradation machine [10] Some regulatory RNAscan also bind specific protein as their target and modifyprotein activity or act by sequestration of substrate Classical

Hindawi Publishing CorporationBioMed Research InternationalVolume 2015 Article ID 153561 16 pageshttpdxdoiorg1011552015153561

2 BioMed Research International

examples of such RNAs include E coli CsrA binding CsrB-CsrC sRNAs and 6S RNA which binds to housekeeping formof RNA polymerase E12059070 [11 12] Tight regulation mediatedby sRNAs is often the result of interconnected circuits actingin feedforward or feedback mechanisms that can regulatetranscriptional factors and two-component systems consti-tuting nodal controls of complex biological networks HenceGram-negative bacteria like Escherichia coli encode over 100sRNAs and several of them have been well characterizedregulating diverse cellular pathways [1ndash3]

In order to achieve high sensitivity many sRNAs aretranscribed in response to specific signals The transcriptionof such sRNAs is often regulated by specialized sigma factorsor by two-component systems In turn sRNAs may providethe feed-back mechanism for the downregulation of sigmafactors and two-component systems once the signal is dealtwith Often downregulation by sRNAs is achieved by reduc-ing the synthesis or translation of these regulatorsThis allowsfor the dampening of elevated stress responses or alteredmetabolic pathways

One of the most well characterized stress responsivesigma factor is RpoE which controls the transcription ofseveral genes with a dedicated function in the envelopebiogenesis (Figure 1) Thus RpoE regulon members in Ecoli include genes encoding periplasmic folding factors (surAfkpA skp and dsbC) proteases [degP ecfE (rseP)] compo-nents of outer membrane proteins (OMPs) assembly (bamA-E) genes whose products are involved in lipopolysaccharide(LPS) translocation and assembly (lptA lptB and lptD)synthesis of phospholipids and lipid A (lpxP lpxD fabZlpxA and lpxB) and the rpoH gene encoding heat shocksigma factor [13] RpoE positively autoregulates its owntranscription and regulates its own activity negatively bytranscribing genes (rseA and rseB) whose products act asnegative regulators [14ndash16] RpoE regulon also includes threesRNAs (RybB MicA and SlrA) [9 17ndash22]

Induction of RpoEupon envelope stress or due to deletionof antisigma factor RseA leads to pronounced reduction inthe amounts ofOMPs Lpp lipoprotein and alterations in LPScomposition [9 22ndash25] The repression of OMPs synthesisoccurs at the posttranscriptional level wherein base-paringsRNAs like MicA inhibits translation and decay of the ompAmRNA and RybB inhibits translation of ompC ompW andompN mRNAs [17ndash19] This inhibition in the synthesis ofmajor OMPs under stress conditions is to maintain an enve-lope homeostasis as some of these OMPs constitute majorabundant proteins under normal growth conditions ThusRpoE-regulated sRNAs MicA RybB and SlrA provide oneof the best examples of the positive regulation (feedforwardmechanism) byRpoE in response to envelope stress andRpoEdownregulation by these sRNAs in a feedback mechanism(Figure 1) [9 24] The orchestrated activities of RpoE MicARybB and SlrA insure homeostatic control of outer mem-brane components such as LPS most abundant lipoproteinLpp and OMPs Such a regulation also allows integration ofdiverse signals from two-component systems with the RpoEsigma factorThere exists an in-built link between OMP con-tent and LPS structure Severe defects in LPS trigger massive

induction of RpoE and this can simply explain reduction inthe amounts of OMPs due to their downregulation of synthe-sis by MicA- and RybB-mediated translational repression

The outer membrane of Gram-negative bacteria is com-posed of highly abundant OMPs and LPS Approximately 2 times106 molecules of LPS cover nearly 75 of the cell surface[26]Thus cellular regulatory controls are in place tomonitorthe biogenesis of OMPs and LPS In many Gram-negativebacteria sRNAs have been shown to play an important rolefor the homeostasis of the envelope In this review specificsRNAs encoded in the E coli genome that regulate thesynthesis composition and structural modification of LPSan essential component of the cell envelope are highlightedAs will be evident specific sRNAs control events in LPSbiosynthesis from its early steps regulating a balance betweenamounts of fatty acids (phospholipids) and LPS as well asincorporation of specific nonstoichiometric modifications inLPS These structural alterations are critical for impartingresistance to antibiotics and survival under defined envi-ronmental niches (Table 1) Dedicated sRNAs most notablycontrol the accumulation of specific LPS glycoforms therebycontrolling LPS composition and its heterogeneity (Figures 12 and 5) The ability to generate glycoforms with truncationsin the outer core of LPS ismodulated by sRNAs andRpoE andis critical for the addition of O-antigen that confers serumresistance [25] In pathogenic Gram-negative bacteria LPSis known to be a major virulence factor Consistent withthe key role of LPS in bacterial virulence the attenuatedvirulence phenotype of hfq mutants can be ascribed toLPS alterations that are regulated by Hfq-dependent sRNAs[1 27]

2 LPS Biosynthesis and Its Heterogeneity

The cytoplasm of Gram-negative bacteria such as E coli issurrounded by an inner membrane (IM) that separates anaqueous periplasmic compartment containing peptidoglycanfrom the outer membrane (OM) The OM is asymmetric innature with phospholipids facing the inner leaflet and theLPS facing towards the outside LPS is a complex glycolipidand constitutes themajor amphiphilic component of theOMIts chemical composition contributes to the permeabilitybarrier function The synthesis of LPS and phospholipidsis tightly coregulated and held at a nearly constant ratio of015 to 10 [26] However LPS is highly heterogeneous incomposition and comprised of a mixture of different glyco-forms [25 28 29] The ratio of different glycoforms whichdiffer due to nonstoichiometric substitutions is regulatedby various stress-responsive two-component systems such asPhoBR (sensing phosphate concentration) PhoPQ (sensingdivalent cations like Mg2+) and BasSR (responsive to Fe3+Zn2+ antimicrobial peptides) and above all by the sigmafactor RpoE the master regulator of envelope biogenesisprocesses [25] Embedded within these regulatory systemsare several noncoding sRNAs (MicA RybB SlrA MgrR andArcZ) that in turn regulate the incorporation of some of thesenonstoichiometric modifications and hence control the LPSheterogeneity (Figures 1 and 2)

BioMed Research International 3

RybB

RseA

LptBLptD

LPS translocationLptA

FtsH

LpxCWaaA

waaQ operon transcription of corebiosynthetic genes

WaaAWaaCWaaF

WaaOWaaB

WaaG

RfaH

WaaR

FabZPhoPQ

PhoBR

WaaHUgd

BasSR

PmrR

LpxT

EptC

Rcs RprA RpoS

LpxPC161

Lipid Amodifications

WaaR

MgrREptB

WaaZ

ppGpp

PagPC160

Defective LPS

KdoII modificationby P-EtN

ArcZGcvB

Glycoformswitches SlrA

Lpp

PG

Sugarnucleotideprecursor

GalK

Spot42

LPSLapB

assembly

GadY

MicA

CsrB

MicA

Coremodifications

modificationsKdo2-lipid A

HO

HO OHO

O OOH

HOO

O OHOH

HOHO

HO

HO

HOO

O

O

OOOO

O OOH

HO

HO

OOH

O

OO

OHOHP

P

NHNH

141414

14

HO

HO

HO

HOO

O

O

OOOO

O OO

HO

HO

OOH

O

OO

OHOHP

P

NHNH

141414

14

HO

120590E

12059032

l-Ara4N

l-Ara4N

Figure 1 Regulatory networks controlling the LPS biosynthesis and nonstoichiometric modifications of lipid A and the LPS core The keyrole mediated by RpoE-regulated noncoding sRNAsMicA RybB and SlrA with their major targets and how RpoE responds to LPS defects isdepicted RNApolymerase in complex with RpoE also transcribes some of the lpt genes whose products are involved in the LPS translocationRybB plays the major role in controlling the LPS composition by the translational repression of WaaR in RpoE-inducing conditions RpoEin turn is subjected to a negative feedback regulation by RybB and SlrA that downregulate the synthesis of major cell envelope componentsto achieve homeostasis RpoE also transcribes the eptB gene leading to the modification of KdoII by P-EtN The translation of eptB mRNAis repressed by PhoPQ-regulated MgrR sRNA MicA and GcvB by base-paring repress the phoP mRNA translation MicA substrate alsoincludes lpxT whose product mediates phosphorylation of lipid A generating triphosphorylated lipid A Central roles played by BasSR-dependent lipid A modifications and PhoBR-dependent GlcUA and P-EtN incorporation in regulating the core biosynthesis are indicatedOn the right side proteolytic regulation of the first committed step in the LPS biosynthesis mediated by the RpoH-dependent FtsHLapBcomplex is presented LapB also couples the LPS synthesis and assembly with translocation system Translation repression of lpp by SlrAregulates the availability of fatty acid pools for the synthesis of phospholipids Spot42 sRNA regulates the availability of sugar nucleotideprecursors for glycosyltransferases in response to the presence of either galactose or glucose by inhibiting the translation of galK within thegal operon

In spite of this heterogeneity LPS in general shares acommon architecture composed of a membrane-anchoredphosphorylated and acylated 120573(1 rarr 6)-linked GlcN disac-charide termed lipid A to which a carbohydrate moiety ofvarying size is attached [30] The latter may be divided into aproximal core oligosaccharide and in smooth-type bacteriaa distal O-antigen

The biosynthesis and translocation of LPS requires thefunction of more than 50 genes Several of them are essentialand unique to bacteria and hence they are excellent targets foridentification of new inhibitors and the development of novelantibiotics Biosynthesis of LPS is thought to occur on the cisside of the plasma membrane as some of the enzymes likethose involved in the early steps of lipid A biosynthesis are


Table 1 Known and predicted sRNAs involved in LPS biosynthesis and its modifications

sRNA Target Effect on LPS Reference(s)

GlmYGlmZ GlmS GlcN6P precursor of LPS [35 39 40]MicA Repression of PhoPQ P-EtN addition to the second Kdo [21 25]

Repression of WaaR Switch between glycoforms IV and V [25]Repression of LpxT Incorporation of lipid A modifications [24]

GcvB Repression of PhoPQ Direct effect on LPS not known [68]

RybB Repression of WaaRMajor control of the synthesis of glycoforms withtruncation in the outer core and incorporation of thethird Kdo

[25]

SlrA Repression of Lpp Levels of fatty acid (phospholipids vs LPS) control [9 22]MgrR Repression of EptB Repression of P-EtN incorporation on the second Kdo [25 56]ArcZ Repression of EptB Impact on LPS not known [57]MicF LpxR Deacylation of lipid A [69]

GadY waaQ operon Overexpression causes transcriptional reduction directeffect on transcription duo to base-pairing not know

[70]

RyeA LpxM Predicted from microarray data [71]RdlA EptA Predicted from microarray data [71]RyjB RfaH Predicted from microarray data [71]

either integral IM proteins (LpxK WaaA LpxL and LpxM)or peripheral IM-associated (LpxB and LpxH) or unstablecytoplasmic protein LpxC [30]

The enzymatic activities of glucosamine-6-phosphatesynthase GlmS and the deacylase LpxC control the bi-osynthetic branch points of major cell envelope compo-nents using two key precursors molecules UDP-N-acetyl-d-glucosamine (UDP-GlcNAc) and R-3-hydroxymyristoyl acylcarrier protein (R-3-hydroxymyristoyl-ACP) respectively[30 31] (Figure 2) Thus the regulation of GlmS and LpxC isimportant and essential for bacterial cell envelope biogenesisIn these processes specific sRNAs play key regulatory roles(Table 1) GlcN6P and its downstream product serve as theglucosamine source for the synthesis of peptidoglycan lipidA enterobacterial common antigen (ECA) and colanic acid(M-antigen) (Figures 2 and 3) ECA and M-antigen can beligated to LPS by the WaaL ligase in the place of O-antigen[32 33]

3 Homologous GlmY and GlmZsRNAs Regulate the Synthesis of GlmSRequired for the Biosynthesis of LPSPrecursor UDP-GlcNAc

GlmS catalyzes the first step in hexosamine metabolismconverting fructose-6-phosphate (Fru6P) into glucosamine-6-phosphate (GlcN6P) which constitutes the first rate-limiting step in the synthesis of UDP-GlcNAc and hence inthe LPS biosynthesis as well as in the other above mentionedconstituents of the cell envelope (Figures 2 and 3) Theconversion of GlcN6P into UDP-GlcNAc requires twoadditional essential enzymes GlmM and GlmU [34] GlmS

synthesis is negatively regulated by its product GlcN6P(feedback inhibition) in a posttranscriptional manner[35]

In E coli glmU and glmS genes are transcribed as anoperon and the corresponding primary transcript is cleavedby RNase E at the glmU stop codon UGA rendering glmUtranscripts unstable The glmSmRNA is also unstable unlessits translation is activated by base-pairing with GlmZ sRNA[36] Specifically GlmZ activates translation of glmS throughan anti-antisense mechanism (reviewed in [37 38]) GlmZacts on the glmS mRNA whose ribosome-binding site isnormally sequestered within a hairpin In concert with theRNA chaperone Hfq GlmZ base-pairs with glmS transcriptand facilitates its translation by stabilizing glmS messageand preventing the formation of inhibitory structure thatoccludes ribosome-binding site of glmS (Figure 3) [39]The activity of GlmZ is controlled by its processing dueto which it can exist in two forms the unprocessed formthat activates glmS translation while the processed form isunable to bind the glmS mRNA (Figure 3) The processedshorter form of GlmZ lacks the glmS target site [36] GlmZand GlmY are structurally homologous sRNA HoweverGlmY lacks the complementarity region to the glmS mRNAand stimulates GlmS synthesis by suppressing the GlmZdegradation by RNase E and RapZ (YhbJ) Thus becauseof the structural similarity with GlmZ GlmY functionsby molecular mimicry and high levels of GlmY titrateRapZ from GlmZ and prevent its processing [38] How-ever GlmZ processing is coupled with GlcN6P levels (seebelow)

In summary it has been shown that the accumulationof GlmS is negatively regulated by RapZ and positivelyby two homologous sRNAs GlmY and GlmZ [35 36 38]


galK

Spot42

CRP

cAMP

Glucose uptake

SgrS

LPS

prec

urso

rs

Export of LPSto OM

Deacylation

Acylation with C16

LpxR

MicF

lpxR

pagP

PhoP

MicA

PagP

Flipping of LPSacross IM

pmrR

BasSR

PmrR

LpxT

lpxT

Posttranslational regulation

O

OH

HOHO

OONH

UDP

LpxA

14

OOH

HO

O ONH UDP

O

HO

O

LpxC

FabZ O

ACP

FabIPhospholipids

PE

PG

UDP-GlcNAc

SlrA Lpp

Core biosynthesis

P-EtN onthe second

Kdo

eptBArcZ

MicAPhoPQ

P-EtN

GcvBMgrR

lpxT waaRGlcIII

MicA RybBWaaGWaaOWaaBWaaR

Transcriptional and translational regulation

GlmZY

waaZpmrD

BasSRglmS

waaB waaS

ACP

OH

ACP

14

OOH

HO

O UDP

O

HO

O

UDP-GlcUDP-Gal

Proteolytic controlby FtsHLapB

arnTeptA

Glycoforms with the third Kdo

LPS translocation

CH3-(CH2)10

CH3-(CH2)10 CH3-(CH2)10

NH2

Kdo2-lipid A

R-3-OH-C14-ACP

trans-2-enoyl-C14-ACP

O

Modificationof 1998400-phosphate

l-Ara4N

O

Figure 2 A flow chart depicting various steps regulated by noncoding sRNAs during the biosynthesis of LPS or its regulatednonstoichiometric modifications The LPS biosynthesis begins with the GlmS-mediated synthesis of GlcN6P GlcN6P serves as a precursorfor UDP-GlcNAc which is a metabolic intermediate for the LPS synthesis UDP-GlcNAc is acylated by LpxA using R-3-hydroxymyristatefollowed by deacylation by LpxCThe expression of the glmSmRNA is regulated by GlmZY sRNAs at the posttranscriptional level while theamount of LpxC is regulated by FtsHLapB-mediated turnover The balanced synthesis of LPS and phospholipids requires SlrA-dependenttranslational repression of Lpp to feed more fatty acids pools for the phospholipid synthesis Kdo

2-lipid A modifications are regulated by

transcriptional induction of the BasSR two-component system and RpoE-dependent induction of the eptB gene PhoPQ activation canamplify induction of the transcription of BasSR-regulated eptA arnT and pmrD genes The PhoP synthesis is negatively regulated byMicA and GcvB sRNAs at the translational level The eptB subjected to translational repression by PhoPQ-regulated MgrR The LPS corebiosynthesis could be further fine-tuned by regulating sugar uptake and the amount of UDP-Gal andUDP-Glc precursors requiring SgrS andSpot42 sRNAsThe RpoE-dependent induction of RybB leads to the synthesis of LPS glycoforms with a third Kdo and truncation of the outercore due to translational repression of WaaR by RybB After the completion of LPS synthesis and its flipping across the IM lipid A can bemodified by LpxT whose activity is repressed at the posttranslational level by PmrR After the incorporation of LPS in the outer membranelipid A may be further acylated by posttranslational activation of PagP or deacylated by LpxR The LpxR synthesis is inhibited by MicF at itstranslational level by base-paring with the lpxRmRNA Note that LpxR is absent in E coli K-12 but is presented in several pathogenic E colistrains and Salmonella Other lipid A modifications not observed in E coli K-12 are not shown

Mutations causing defects in RapZ or increase in GlmY orGlmZ concentrations lead to increased expression of glmSEach of these sRNAs has a unique role and they work ina hierarchical feedback loop to activate glmS expression inresponse to intracellular levels of GlcN6P (Figure 3) [3638] Under limiting GlcN6P conditions homologous GlmY

sRNA accumulates and sequesters RNase adaptor proteinRapZ preventing GlmZ processing [35 40] In contrast athigh concentrations of GlcN6P GlmZ is preferably boundby RapZ and consequently degraded by RNase E (Figure 3)GlmY does not bind Hfq and hence GlmY functions asa molecular decoy acting as an antiadaptor titrating away


AdaptorRapZ

AdaptorRapZ

AdaptorRapZ

AdaptorRapZ

glmUglmS operon glmUglmS mRNA

Free glmS mRNAStabilization

SD

GlmS

GlcN6P UDP-GlcNAc

Lipopolysaccharide(LPS)

Peptidoglycan

Enterobacterialcommon antigen

(ECA)Colanic acid(M-antigen)

Translation

Hfq

glmS mRNA

Complex

Processing

GlmZ

GlmY

DegradationglmU mRNA

glmS mRNA

RNase E

ProcessingglmS mRNA

SD Translation

Degradation

Complex

Processing

RNase E

UGA

GlmZ GlmZ

GlmY

GlmZ

GlmY

GlmZ GlmZ

GlmY

Free

Transcription

LowGlcN6P

HighGlcN6P

(a)

(b)

(c)

RapZ

RapZ

Figure 3 Hierarchical regulation of glucosoamine-6-phosphate synthase GlmS (a) Genes glmUS constitute a bicistronic operon which iscleaved by RNase E at the UGA stop codon of glmU generating into monocistronic mRNAs that are rapidly degraded (b) Under GlcN6Plimiting conditions the glmS mRNA base-pairs with intact GlmZ leading to stabilization of the glmS mRNA and also activation of itstranslation by disrupting inhibitory stem-loop structure that otherwise sequesters the Shine-Dalgarno (SD) This base pairing requires HfqUnder GlcN6P limiting conditions GlmZ processing is prevented by sequestration of the RapZ adaptor protein preventing RapZ targetingto RNase E-mediated degradation of GlmZ (c) When GlcN6P amounts are high GlmY sRNA is present in low amounts This allows therecruitment of RapZ to GlmZ leading to RNase E-mediated cleavage This cleavage leads to the generation of a processed form of glmZthat lacks complementarity to glmS and hence inability to activate glmS translation due to inability to access SD This also leads to a rapiddegradation of the glmSmRNA

GlmZ degradation system of the RNase E adaptor RapZ[36] Hence the synthesis of GlcN6P is tightly regulated inresponse to its synthesis demands and concentration sensingby GlmYGlmZ sRNAs Thus it constitutes an early step inthe synthesis of LPS precursor UDP-GlcNAc

An interesting connection in the regulation of the tran-scription of rapZ and glmY with RpoE activation is likely andneeds further studies The rapZ gene is located within therpoN operonThe transcription of the rpoN gene is positivelyregulated by RpoE and can also have an impact on its mRNA

levels from RpoE-dependent transcription regulation of pro-moters located upstream of lptAB genes [41] FurthermoreRpoN and the two-component system QseFE regulate thetranscription ofGlmY Interestingly QseFE and its orthologsplay critical roles in virulence of several enterobacterialpathogens like Salmonella Yersinia pseudotuberculosis andenterohemorrhagic E coli (EHEC) [42] Thus these findingsfurther underline the importance of sRNAs involvement likeGlmYS in other regulatory pathways that could impact notonly LPS but also other virulence factors


4 Balanced Biosynthesis ofLPS and Phospholipids and FeedbackControl by sRNAs

41 Feedback Control by RpoE-Dependent SlrA sRNA Thebiosynthesis of LPS beginswith the acylation ofUDP-GlcNAcwith R-3-hydroxymyristate derived from R-3-hydroxymyris-toyl-ACP [43] R-3-Hydroxymyristoyl-ACP also serves as aprecursor for the synthesis of phospholipids [44]The secondreaction of the lipid A biosynthesis is catalyzed by LpxC[UDP-3-O-(R-3-hydroxymyristoyl)-N-acetylglucosaminedeacetylase] constituting the first committed step in the LPSsynthesis as the equilibrium constant for the first reactioncatalyzed by LpxA is unfavourable Next seven additionalenzymes are required for the completion of synthesis ofthe minimal LPS structure Kdo

2-lipid A which acts as a

substrate for further sequential incorporation of varioussugars by specific glycosyltransferases (Figure 2)

The fabZ gene encodes the R-3-hydroxymyristoyl-ACPdehydratase which catalyzes the first key step in the phospho-lipid biosynthesis Thus the fundamental common substrateof LpxC and FabZ R-3-hydroxymyristoyl-ACP comprises anessential branch point in the biosynthesis of phospholipidsand the lipid A part of LPS (Figure 2) Hence an in vivocompetition of FabZ and LpxC for their common substratesets a balance in the synthesis of phospholipids and lipid A[45] High level of the LpxC accumulation is toxic to cellsdue to the excess of LPS over phospholipids and resultingdepletion of the common substrate R-3-hydroxymyristoyl-ACP [9 46] Thus regulation of LpxC amounts is crucialfor this critical biosynthetic checkpoint LpxC is an unstableprotein and its turnover is mediated by the essential IM-located Zn-dependent metalloprotease FtsH in conjunctionwith the recently identified heat shock protein LapB [9 4647] LapB is also an IM-anchored protein and contains TPRrepeats in its N-terminal domain and a rubredoxin-like C-terminal domain both of whichwere found to be essential foractivity [9]The absence of either LapB or FtsH is toxic as cellsaccumulate elevated levels of LPS with a phenomenon thatcan be compensated by suppressormutations in the fabZ geneor by overexpression of fabZ which restore a balance betweenphospholipids and LPS [9 46 47] Alternatively increasingthe free fatty acid pools that serve as precursors for thephospholipid synthesis can also overcome the lethality in theabsence of LapB (Figures 1 and 2) [9] This finding was basedon the isolation of multicopy suppressor identifying a newsRNA SlrA [9] The SlrA-mediated multicopy suppressioncould be attributed to lpp (encoding Braunrsquos lipoprotein) asa substrate of SlrA sRNA (Figures 1 and 2) Consistent withsuch a notion transposon insertion in the lpp gene alsorestored lapB defects [9] Thus reduction in Lpp mediatedby overexpression of SlrA sRNA or loss of lpp can bypasslethality when the LPS synthesis is increased due to accumu-lation of LpxC Lpp is themost abundant lipoprotein in E coli(7 times 105 molecules per cell) and contains three lipid chainsHence its depletion increases the pool of free fatty acidsfor the synthesis of phospholipids and helps to restore thebalance with LPS Indeed the overexpression of SlrA sRNAalso called MicL leads to repression of the Lpp synthesis and

hence mimics a loss of function mutation in the lpp gene[9 22] SlrAMicL was shown to inhibit lpp at its translationallevel by direct base-pairing in an Hfq-dependent manner

slrAwith its own promoter is encodedwithin the 31015840 end ofthe coding region of the cutC gene [9 22] SlrA was found tobe an 80 nt sRNA which is synthesized as a 307 nt precursormRNA that is processed At present the enzyme requiredfor the processing of slrA mRNA is unknown Interestinglytranscription of slrA is directed by E120590E polymerase andthus slrA is the third known sRNA of the RpoE regulon[9 22] SlrA expression also provides a feedback mechanismof control of RpoE activity since overexpression of SlrAsRNA downregulates elevated envelope stress responses bymonitoring Lpp amounts [9] Indeed overexpression of SlrAcauses downregulation of DegP synthesis in (lapA lapB)mutants that is otherwise elevated in such backgrounds dueto the RpoE induction [9] Thus repression of the lppmRNAtranslation leading to restoration of phospholipid synthesisto counteract increase in LPS amount and the control ofRpoE envelope stress response by a feedback mechanism isthe main function of SlrA

42 A Novel cis-Encoded sRNAMay Control LpxC Amounts inPseudomonas aeruginosa Interestingly SlrA sRNAandLapBare absent in Pseudomonas aeruginosa [9] LpxC of P aerugi-nosa is not a substrate of FtsH [48] Examination of sRNAs inP aeruginosa revealed the presence of an 84 nt sRNAPA4406located in the intergenic region between ftsZ and lpxC genesThe existence of this sRNA has been verified experimentallyand it is transcribed in the same sense as its potential targetlpxC [49ndash51] It is likely that this sRNA controls LpxC basedon the repeated isolation of amutation that confers resistanceto novel LpxC inhibitor LpxC-4 [51 52] This mutation (C toA change) is located 11 nt upstream of the lpxC translationalstart site and maps to the 31015840 end of the sRNA PA4406 [51]Predicted structure suggests that this C to A mutation islocated within the hairpin structure that pairs with G 18nucleotide upstream of lpxC and could impact accessibility ofribosome-binding siteThismutation causes a 3-fold increasein lpxC expression which could be as a result of increasein either the mRNA level or its differential turnover Theexistence of such regulatory sRNA reflects an additionalrole for RNAs in this important pathogenic bacterium inregulating LpxC amounts and hence the LPS biosynthesisThe location of this cis-acting 84 nt sRNA in P aeruginosa inthe same sense as its putative target is rather rare Most of thecis-acting sRNAs are located on the opposite strand of theirtarget [1ndash3] Further most of the LPS regulating sRNAs likeMicARybB SlrA andMgrR are trans-acting thus providinganother interesting dimension to this PA4406 sRNA in Paeruginosa However it is not clear if PA4406 sRNAarises dueto transcriptional processing or if it has its own promoter

5 Biosynthesis of LPS andIts Regulated Modifications

Lipid A and Kdo are the most conserved elements of LPSbetween species In E coli a bisphosphorylated lipid A


precursor molecule termed lipid IVA is synthesized fromUDP-GlcNAc following six distinct enzymatic reactionsThelipid IVA precursor serves as an acceptor for the additionof two Kdo residues mediated by the bifunctional enzymeKdo transferase WaaA This reaction results in the synthe-sis of the Kdo

2-lipid IVA intermediate This intermediate

serves as a substrate for late acylation reactions catalyzedby LpxL (lauroyl transferase) and LpxM (myristoyl trans-ferase) to generate hexa-acylated lipid A (Kdo

2-lipid A) and

also for various glycosyltransferases for the completion ofcore biosynthesis However at low temperature (12∘C) LpxPmediates the addition of palmitoleate chain (C161) in sim80of LPS molecules in the same place where lauroyl residueis usually present [53] The lpxP gene is RpoE-regulatedat the transcriptional level (Figure 1) The incorporation ofpalmitoleate in place of laurate suggests the presence ofsuch unsaturated fatty acids aids in homeoviscous adaptationin response to cold stress or when LPS is composed ofglycosylation free lipid IVA derivatives as observed in waaAmutants [54]

The Kdo2-lipid A constitutes the minimal LPS structure

that is required for viability of bacteria like E coli understandard laboratory growth conditions [54] Interestinglythis structure is quite dynamic and is often subjected tononstoichiometric modifications that involve activation oftwo-component systems and specific noncoding sRNAs (Fig-ure 4) Some of these modifications are critical for resistanceto cationic antimicrobial peptides and also for bacterialvirulence in pathogenic bacteria Most of the modificationsinvolve either reducing the net negative charges of lipid Aby modifying the 1 and 41015840 ends of phosphate residues orthe addition or removal of acyl chains (Figure 4) Similarlymodifications are also known to occur in the core regionlike incorporation of phosphoethanolamine (P-EtN) uronicacids or additional Kdo residues [25 28]

Genes involved in the synthesis and modifications oflipid A and LPS are regulated by several transcriptional andposttranscriptional factors including RpoE sRNAs that maybe or not regulated by RpoE and the two-component systemsBasSR (also known as PmrAB) PhoPQ and PhoBRin E coli and Salmonella (Figures 1 and 2) Additionaltwo-component systems like RcsBC EvgAS may furthermodulate one or more of these systems or induce a specificgene that is subjected to multiple transcriptional controls forexample the ugd gene [28 55] It is known that RpoE regulonmembers include genes whose products are involved in LPSbiosynthesis LPS transport like lpt genes [13 41] and LPSmodification system controlled by the product of the eptBgene and MicA and RybB sRNAs [24 25 54 56 57] (Figures1 and 2) MicA at the same time negatively regulates the two-component system PhoPQ by the translational repressionof PhoP [21] To integrate signal transduction and crosstalk between two-component systems and RpoE-dependentsRNAs severe defects in LPS dramatically induce RpoEactivity and this in turn is reflected in LPS composition andits modifications [25 54]

In E coli and Salmonella enterica serovar TyphimuriumBasSR (PmrAB) is induced upon exposure to low pHexcess of Fe3+ Zn2+ and Al3+ challenge by antimicrobial

peptides or treatment with the nonspecific phosphataseinhibitor ammoniummetavanadate [28 54 58ndash60]Themostnoticeable changes in lipid A involve the nonstoichiometricincorporation of P-EtN and 4-amino-4-deoxy-l-arabinose(l-Ara4N) residues by EptA and ArnT respectively (Fig-ure 4) EptA and ArnT transferases mediate the modificationof 1-phosphate and 41015840-phosphate by P-EtN and l-Ara4Nrespectively Covalent modifications by l-Ara4N and P-EtN cause decrease in the overall negative charge which isessential for resistance to cationic antimicrobial peptides andalso for the outer membrane integrity [60 61] eptA andarnT genes belong to the BasSR (PmrAB) regulon In 119878Typhimurium the activation of PhoPQ upon depletion ofMg2+ and Ca2+ also leads to PmrAB induction and thusallows integration of signals from different environmentalcues This cross talk between PmrAB and PhoPQ systemsrequires the adaptor protein PmrD [62] The activation ofthe PhoPQ system also induces transcription of pagP andpagL and hence upregulation of the encoded proteins whichacylate and deacylate lipid A respectively [61] (Figure 4)However PagP and PagL modification occurs posttransla-tionally after LPS is incorporated in the OM Recently ithas been shown that even in E coli PmrD can link thePhoPQ and BasSR two-component systems upon depletionof Mg2+ and promote lipid A modification by inducting thetranscription of eptA and arnT genes [63]

Activation of BasSR also induces the transcription ofGcvB sRNA [64] GcvB is relatively highly conserved Hfq-dependent sRNA It is the one of the most globally actingposttranscriptional regulators in bacteria potentially regulat-ing sim1 of all mRNAs in Salmonella and E coli [65ndash67] Itsregulon is highly enriched with transporters of amino acidsand short peptides including the major ABC transportersDpp and Opp Its regulon members also include amino acidbiosynthesis proteins and major transcription factors such asLrp and PhoP As PhoP is involved in lipid A modificationstranslational repression of PhoP byGcvB [68] adds additionaldimension to diverse inputs in modulating lipid A and LPScore modifications that could fine tune BasSR and PhoPQinduction In E coli a deletion derivative of the gcvB strainwas found to have increased expression of genes involvedin the O-antigen biosynthesis like rfbAC wbbH wbbKand wbbJ [65] Thus MicA- and GcvB-dependent PhoPrepression could jointly control lipidA andLPSmodificationsand given the conservation of these sRNAs they couldplay important roles in altering LPS and hence virulencephenotype

6 Modification of Lipid A Role of MgrR andMicA sRNAs

Several sRNAs play important modulatory roles for themodification systems of lipid A For example activation ofPhoPQ induces the transcription of the mgrR sRNA MgrRhas been shown to repress translation of the eptBmRNA [56]The eptB gene is positively regulated at its transcriptionallevel by RpoE [57] EptB transfers P-EtN to the secondKdo in the inner core and also contributes to resistance to


HO

HO

HO

HO

HO

OH

O

O

O

O

OO

OO

O

O

OH

HOO

O

O

O O

O

OH

O

OHO

OH

O

OO

OH

OH

OH

HO

P

P

NH

NH

12

1414

1414

HO

O

14

O

Lipid A

Kdo2

(a)

O

14

HO

HO

HO

OH

O

O

O

O

OO

O O

O

O

OH

HOO

O

OO O

O

OH

O

OO

OHO

O

OO

OOH

R2

OHHO

P

P

NH

NH

12

14

14 1414

HO

HOO

OH

O

R1 O

O

OHP

EptA

OO

OP P

LpxT

RdlA

MicA

OO

OH

EptBMgrR

LpxRMicF

ArcZ

O

16PagP

O

OHOHOH

NH2

NH2

PNH2

(b)

Figure 4 Chemical structures of unmodified Kdo2-lipid A (a) and modified Kdo

2-lipid A derivatives (b) The synthesis of P-EtN transferase

EptB is repressed by PhoPQ-regulated MgrR sRNA MicA and MicF repress LpxT and LpxR respectively by base-pairing of their mRNAsRdlA sRNA is predicted to act on eptA however impact on lipid A modification is not known R1 and R2 indicate modification sites

antimicrobial peptides like polymyxin B [54 56 72] In theintegration of this signal transduction PhoPQ is repressedby MicA sRNA thus linking RpoE and PhoPQ in responseto envelope stress MicA is induced upon RpoE activationand represses phoP mRNA translation [21] MicA base-pairswith the phoP mRNA in its translation initiation region andinhibits its translation by competing for ribosome binding[21] (Figures 2 and 4) PhoP translation is also repressedby base-pairing with GcvB sRNA [68] However this GcvB-mediated repression of phoPmRNA translation does not havean impact on MgrR sRNA [68]

Structural examination of LPS of a strain lacking WaaCheptosyltransferase I revealed that lipid A lacks P-EtN evenunder eptA-inducing conditions but preferentially incorpo-rates P-EtN on the secondKdo in Ca2+-supplemented growth

medium [54] Under these conditions RpoE is induced dueto LPS defects causing induction of the transcription of theeptB gene Ca2+ can repress PhoPQ which turns off thesynthesis of mgrR sRNA whose transcription is PhoPQ-dependent and at the same time allows the synthesis of EptB[56] Ca2+ is also required for the enzymatic activity of EptBand thus enhances the incorporation of P-EtN on the secondKdo [72] Lack of P-EtN in lipid A as occurring in waaCmutants can be explained by the preferred incorporation ofP-EtN on the second Kdo rather than in lipid A presumablyto maintain a homeostatic control on such incorporationHowever in the absence of EptB lipid A of waaC mutantexhibits nonstoichiometric incorporation of P-EtN [54]

Activation of RpoE can override MgrR-mediated inhi-bition of eptB expression and hence promote incorporation


Lipid A

2

4

WaaAWaaQ7

1

PWaaP

44

PWaaY

5 KdoI 2

KdoII

WaaA

HepIII

HepI 13HepII 13GlcI 13

WaaG

6

1WaaU

HepIV

GlcIII 1

WaaR

WaaB6

1

Gal

Glycoform I

WaaCGlcII 12

WaaO WaaF

(a)

Lipid A

2

4

PEptC

P-EtN

1

6

WaaSRha

2

4

WaaZ

Glycoform IV

GlcII 1 GlcI 13 HepII 13

Gal

HepI 1 53 KdoI 2

KdoII 5

1

7

44

P

MgrR

ArcZEptB

RybBWaaRRpoE

KdoIII

HepIII 1

RpoE

Hfq

MicAlowast

(b)

Lipid A

2

4

PEptC

P-EtN

1

6

EptB

2

4

WaaZ

Glycoform V


Gal

HepI 1 53 KdoI 2

KdoII 7

1

7

1

WaaSRha5

P-EtN

44

P

KdoIII

HepIII

RybBWaaRRpoE

RpoE

Hfq

RpoE

MicAlowast

(c)

Lipid A

2

4

PEptC

P-EtN

1

6

EptB

2

4

WaaZ


Gal HepIII

HepI 13 5 KdoI 2

KdoII 7

1

7

1

WaaSRha5

P-EtN

4

WaaH7

1

GlcUA

Glycoform VII

KdoIII

RybBWaaRRpoE

RpoE

HfqRpoE

MicAlowast

(d)

Figure 5 Schematic depiction of various LPS glycoforms observed in E coli K-12 Glycoform I is the major LPS glycoform under nonstresscondition that is in the absence of RpoE induction and other two-component systems (a) Induction of RpoE leads to the accumulation ofglycoforms IV V andVII due to RybB- andMicA-mediated translational suppression ofWaaR and induction ofwaaZ andwaaS transcriptionthat may also involve Hfq In this process RybB plays the central role Glycoforms IV V and VII contain a third Kdo and rhamnose witha concomitant truncation in the outer core (b c and d) Glycoform IV accumulates when MgrR repression of eptB is predominant Thetranslation of the eptB mRNA is repressed by base-paring with MgrR under PhoPQ-inducing conditions and also by ArcZ sRNA (b)When the RpoE induction is maximal RpoE-driven transcription overrides MgrR-mediated repression of the EptB synthesis due to thehyperinduction of RpoE-regulated transcription of the eptB gene Under such conditions RybB at the same time represses translationally theWaaR synthesis (c)The glycoformVII is the major glycoformwhen RpoE-dependent eptB and rybB expression is induced with simultaneousinduction of the waaH gene transcription leading to the GlcUA incorporation (d)

of P-EtN on Kdo [54 56 57] This can be attributed toMicA-dependent inhibition of PhoP under RpoE-inducingconditions as well as induction of strong RpoE-dependenttranscriptional activation of the eptB gene [21] As we willdiscuss further P-EtN incorporation on the second Kdocan lead to further LPS structural alterations when RpoE isinduced with intact LPS biosynthesis genes (Figure 5) Anadditional layer involving regulation of the eptB expressionhas revealed a role for ArcZ sRNA (Figure 4) Both ArcZand MgrR are Hfq-dependent and inhibit translation andexpression of eptB by base-pairing although in response todifferent environmental cues ArcZ inhibits eptB expressionin an ArcAB-dependent manner whereby phosphorylatedArcA represses ArcZ synthesis when oxygen concentrationsare low [57] Thus EptB is controlled at several levels andexplains its importance in contributing to LPS diversity andresistance to antibiotics like polymyxin B

RpoE-regulated MicA sRNA is predicted to be involvedin controlling phosphorylation of lipid A by regulatingLpxT (Figure 4) MicA can base-pair with the lpxT mRNAand inhibit its translation [24] Although its effect on theLPS structure has not been directly examined structuralanalysis of LPS obtained from several different strains undersimultaneous RpoE- and BasSR-inducing conditions showsthe absence of LpxT-dependent phosphorylation of lipid Apresumably due to the transcriptional induction of micARather under such conditions EptA-dependent P-EtNmodi-fication is observed and accumulation of triphosphorylatedlipid A species does not occur [25] However the inhi-bition of LpxT activity upon BasSR-inducing conditionsis due to the expression of a short peptide PmrR whichhas been shown to directly bind to LpxT [73] and henceconstitutes posttranslational control (Figures 1 and 2) Thusboth BasSR and MicA contribute to the inhibition of LpxT


phosphorylation and allow incorporation of P-EtN andAra4N in lipid A

7 MicF and Regulation of Acylation

MicF sRNA is another regulatory trans-acting base-pairingRNA that controls lipid A modification by promoting thedegradation of lpxRmRNA LpxR is a lipid A deacylase withCa2+-dependent 31015840-O-deacylase activity and such modifiedlipid A is less bioactive [69 74] The lpxR gene is absent inE coli K-12 but is found in the genomes of E coli O157H7Yersinia enterocolitica Helicobacter pylori and Vibrio cholera[74] MicF has been shown to bind to the lpxRmRNA withinits coding region as well as in the ribosome-binding site [69]The base-pairing of MicF within the coding sequence of thelpxRmRNA decreases its stability by rendering it susceptibleto degradation by RNase E [69] In Y enterocolitica howeverlpxR is negatively regulated by the PhoPQ system and theregulator RovA [75] As LpxR-mediated deacylation occursafter LPS translocation the regulation of LpxR by MicFcontributes to LPS modification event that occurs in the OMexpanding the role of sRNAs at various steps of the LPSbiosynthesis and its modification

8 Incorporation of the Third Kdo byWaaZ and Coordinated Repression ofWaaR Synthesis Regulation by RybB andMicA sRNAs

LPS is structurally highly heterogeneous composed of severalglycoforms that differ due to the incorporation of P-EtN onthe second Kdo incorporation of a third Kdo incorporationof additional sugars like rhamnose GlcN uronic acids andalterations in the numbers of phosphate residues in theLPS core In most cases examined the inner core has beenfound to contain an 120572-(2-4)-linked Kdo disaccharide whichis transferred by the bifunctional enzyme WaaA Undernormal laboratory growth conditions the majority of LPSis composed of glycoform I and minor amounts of threeadditional glycoforms II III and IV can also be observed[29] Glycoform I contains two Kdo residues in the inner coreand four heptoses and four hexoses attached in specific orderin the inner core and the outer core (Figure 5) Thus far 7glycoforms have been structurally identified and extensivelycharacterized [28] Under RpoE-inducing conditions LPSis primarily comprised of glycoform V and its derivatives[25] This molecular switch is highly regulated and requiresinduction of the RpoE-transcribed genes eptB sRNAs micAand rybB and the transcriptional upregulation of waaZ witha concomitant repression of WaaR synthesis This switch isfurther amplified upon induction of the BasSR and PhoBRtwo-component systems [25]

Briefly waaZ encodes a Kdo transferase required for theincorporation of the third Kdo [25] The waaS gene encodesthe rhamnosyl transferase and its incorporation requiresprior addition of the third Kdo [25] (Figure 5) The WaaRglycosyltransferase mediates the incorporation of the thirdGlc in the outer core of LPS which subsequently serves as

an acceptor for the addition of the terminal heptose (HepIV)by WaaU (Figure 5) LPS species defining glycoforms IVand V have the same molecular masses but are structurallydifferent Glycoform IV is the most abundant LPS underPhoBR and BasSR induction and when RpoE-regulatedeptB encoding P-EtN transferase is not induced Under suchconditions EptB synthesis is silenced by PhoPQ-dependentMgrR sRNA [56] However when RpoE is induced forexample in the absence of the antisigma factor RseA or inthe absence of Hfq most LPS species are represented byglycoform V which contains EptB-dependent P-EtN on thesecond Kdo [25] P-EtN modification of the second Kdofavors the incorporation of rhamnose on the terminal thirdKdo which is otherwise attached to the second Kdo inglycoform IVThus the preferential synthesis of glycoform Vand its derivatives even when the EptB synthesis is repressedby MgrR sRNA suggests that RpoE induction overcomessilencing of the eptB expression and promotes this switch[25]

It is interesting that the glycoforms containing the thirdKdo and rhamnose such as glycoform IV and V have a con-comitant truncation of the terminal disaccharide as if WaaRis limiting [25] (Figure 5) Among several strains tested fortheir LPS composition the absence of RseA antisigma factorthat leads tomaximal induction of RpoE causes near exclusivesynthesis of glycoform V and VII or their derivatives Themolecular basis of this switch is explained by the inductionof eptB expression and by two additional factors Firstlyunder such conditions the waaZ transcription and WaaZamounts are increased promoting the incorporation of thethird Kdo [25] However the transcription of the waaZ geneis not directly regulated from RpoE-recognized promoterand hence could occur after initiation of transcription inan Hfq-dependent manner In agreement with this notiona 3-fold increase in waaZ mRNA levels has been observedin an hfq mutant [76] Secondly the synthesis of WaaR isrepressed preventing the addition of terminal disaccharidein the outer core which is absent in such LPS structuralforms Thus incorporation of the third Kdo and truncationin the outer core are highly coordinated processes and areorchestrated by RpoE-dependent sRNAs This occurs dueto the simultaneous induction of Hfq-dependent MicA andRybB sRNAs Thus introduction of a deletion of the rybBgene in strains lacking RseA restores the LPS synthesis tonear normal levels and suppresses the synthesis of glycoformsthat incorporate Kdo

3Rha A similar but partial restoration

in LPS composition is also observed in the absence ofMicA in strains lacking RseA This is further supportedby the findings that WaaR amounts are reduced in rseAmutants but restored to the normal levels when RybB issimultaneously removed Consistent with repression ofWaaRsynthesis favoring the incorporation of LPS with the thirdKdo waaR mutants also synthesize LPS derivatives corre-sponding to glycoform IV without any requirement for RpoEinduction

E coli preferentially synthesizes glycoform IV containinga third Kdo with truncation of the outer core disaccharidein PhoBR-inducing conditions without RpoE induction[25] This switch from the usual glycoform I to IV can also


be explained by increased transcription of the rybB sRNA[77] This can provide an explanation based on the RybB-mediated repression of WaaR [25] Thus sRNAs controlthe LPS biosynthesis and its modifications at several levelsRegulation of the third Kdo incorporation is importantsince this event prevents the incorporation of O-antigen asit is attached to the terminal heptose which is absent inglycoforms IV and V Further Kdo residues form importantcontacts that are vital for TLR4-MD2 mediated immuneresponse

Pathogenic bacteria overcome the host defense in avariety of ways including subversion of function of dendriticcells (DC) that can interfere with the innate immune systemDCs express C-type lectin called DC-SIGN with whichseveral pathogenic bacteria and even nonpathogenic E coliinteract through LPS This interaction promotes adherenceand phagocytosis and requires LPS core sugar residues [78]Among several strains of E coli with defects in the LPS corebiogenesis most significantly waaR mutants are resistant tophagocytosis by HeLa-DC-SIGN and the predicted ligand iseitherGlcIII orGlcNAc [78] SinceWaaR is the target of RybBsRNA and GlmYGlmZ are required for synthesis of UDP-GlcNAc this implies that LPS alterations by such sRNA-mediated interaction can play important role in bacterialadhesion and phagocytosis

In summary translational repression by PhoPQ-regulated MgrR of eptB expression and repression of WaaRsynthesis by RybB promotes the synthesis of glycoform IVHowever induction of RpoE leads to increased transcriptionof waaZ eptB and rybB This leads to switch causingsynthesis of glycoform V Further BasSR activation underRpoE-inducing conditions amplifies this incorporation ofglycoforms with a third Kdo and rhamnose due to increasedexpression of waaS Thus RpoE PhoPQ BasSR andPhoBR jointly control LPS composition using a network ofsRNA-mediated control (Figures 1 2 and 5)

9 Impact of Regulated LPS Modifications onVirulence Associated Phenotypes

Deletion of PhoPQ-regulated MgrR results in a 10-foldincrease in the resistance to polymyxin B PhoPQ andPmrAB are both activated following endocytosis of live STyphimurium cells by RAW 2647 macrophage tumour cellsresulting in multiple partial covalent modifications of lipid A[79] Addition of the l-Ara4N and palmitate residues to lipidA confers resistance to polymyxins and 120573-defensins respec-tively [80 81] Additionally competitive infection experi-ments in mouse models of S Typhimurium infection showeda decrease in survival ofmutants unable to incorporate P-EtNwhen compared to wild-type strains [82] RybB-mediatedrepression ofWaaR could as well contribute to bacterial adhe-sion and phagocytosis as discussed earlier Severe defects inLPS or lack of PhoP have been shown to cause defects in col-onization biofilm formation and sensitivity to antimicrobialpeptides in many pathogenic bacteria such as Yersinia pestis[83]

10 Other Targets of sRNAs inLPS Biosynthesis

In Salmonella it has been reported that in the absence of Hfqseveral genes whose products are involved in lipid A (lpxAand lpxD) phospholipids (fabZ) and O-antigen biogenesis(rfbACKMJU) are upregulated [84] It will be interesting toidentify if any specific sRNA is involved in this process SomeLPS alterations have also been reported in an E coli UPECvariant lacking hfq [27] Expression of lpxC ugd and waaHhas been shown to be upregulated alongwith the concomitantupregulation of GcvB RybB and GadY sRNAs followingphosphate starvation [28 77]

Alterations in LPS profiles have been reported in strainslacking CsrA or upon its overexpression [85] CsrA is a RNA-binding protein and was initially identified as a regulatorof glycogen biosynthesis [86] CsrA binds at GGA-richmotifs and could promote RNA decay [87] The activity ofCsrA is regulated by specific sRNAs (CsrBCsrC) Shigellastrains lacking CsrA also have been shown to have alteredLPS profiles [88] Similarly transcriptome analysis of strainlacking YbeYRNase revealed that eptA lpxM and rfaH couldbe regulated by RdlA RyeA and RyjB sRNAs respectively[71] YbeY has been shown to regulate the expression of somesRNAs [71] Further LPS structural analysis will be requiredto draw a direct conclusion of the involvement of theseadditional sRNAs in the control of LPS biosynthesis and itsmodifications In the pathogenic bacterium Porphyromonasgingivalis the synthesis of LPS was found to be altered bydeletion of antisense RNA molecule located within a 77-bpinverted repeat element This element lies in the 51015840 region ofthe K-antigen synthesis locus [89]

Considering the involvement of other sRNAs it is tempt-ing to speculate that depletion of sugar nucleotides poolscould contribute to structural heterogeneity Thus Spot42-dependent repression ofGalK could limit precursorUDP-Glcand UDP-Gal availability when CRP is activated [90] Thereis precedence for such a signal transduction conferring phe-notype that can be attributed to reduction in the availabilityof UDP-Glc pools A derivative of enterohemorrhagic E coliO157H7 with lpxMmutation was found to have a truncationin LPS due to limiting amounts of UDP-Glc precursor Thisdefect could be complemented by the overexpression of galU[91] A similar scenario can be imagined if the synthesisof sugar transports is impaired which require SgrS sRNAsRNA-dependent LPS heterogeneity and truncation of LPShas also been shown when GadY sRNA is overexpressed in awaaYmutant while studying role of LPS in biofilm formation[70] This truncation has been ascribed to reduction in theexpression of waaQ operon Further work will be required toknow exact molecular basis of GadY control of expression ofthe waaQ operon

11 Conclusion

It is now abundantly clear that sRNAs play important roles ingene expression and regulate several functions Until recentlythe main function of MicA and RybB sRNA was thought


to be translational repression of OMP biosynthesis Bothof these sRNAs are regulated by the RpoE sigma factorRpoE controls the expression of genes involved in the cellenvelope biogenesis in response to changes in OMP andLPS composition Here we have highlighted that MicA andRybB also control the LPS biogenesis and its modificationsFurther the newly identified RpoE-regulated sRNA SlrAcontrols the expression of the most abundant protein Lppand negatively regulates RpoE expression Together thesethree sRNAs monitor LPS and the OM composition As LPSis highly heterogeneous and additional sRNAs have beenpredicted to regulate the LPS biosynthesis or its modifica-tions it will be interesting to examine LPS composition ofstrains lacking these sRNAs or when they are overexpressedAlso it will be pertinent to examine how RybB mediatesthe translational repression of WaaR an enzyme that iskey to the synthesis of different glycoforms The chemicalstructure of such novel glycoforms should be resolved andtheir inability to elicit or escape immune response shouldbe considered carefully In addition to the PhoPQ andBasSR two-component systems PhoBR also regulates theincorporation of glucuronic acid and P-EtN in the inner coreMicroarray studies have revealed that expression of severalsRNAs is also altered by the PhoBR-regulated response uponphosphate starvation Further research is required to addressif these PhoBR-regulated sRNAs impact the structure andcomposition of LPS In summary we can conclude thatseveral sRNAs regulate LPS composition and contribute to itsstructural diversity at several steps

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

The authors gratefully acknowledge D Missiakas for helpfulsuggestions and D Biernacka for help during the revisionThis work was supported by National Science Grants 201103BNZ102825 and 201311BNZ202641 to Satish Raina

References

[1] K Papenfort and J Vogel ldquoSmall RNA functions in carbonmetabolism and virulence of enteric pathogensrdquo Frontiers inCellular and Infection Microbiology vol 4 article 91 2014

[2] CMichauxNVerneuil AHartke and J-CGiard ldquoPhysiolog-ical roles of small RNAmoleculesrdquoMicrobiology vol 160 no 6pp 1007ndash1019 2014

[3] G Storz J Vogel and K M Wassarman ldquoRegulation by smallRNAs in bacteria expanding frontiersrdquoMolecular Cell vol 43no 6 pp 880ndash891 2011

[4] R R Breaker ldquoProspects for riboswitch discovery and analysisrdquoMolecular Cell vol 43 no 6 pp 867ndash879 2011

[5] M T Morita Y Tanaka T S Kodama Y Kyogoku H Yanagiand T Yura ldquoTranslational induction of heat shock transcrip-tion factor 12058932 evidence for a built-in RNA thermosensorrdquoGenes amp Development vol 13 no 6 pp 655ndash665 1999

[6] J Kortmann and F Narberhaus ldquoBacterial RNA thermometersmolecular zippers and switchesrdquo Nature Reviews Microbiologyvol 10 no 4 pp 255ndash265 2012

[7] L Argaman R Hershberg J Vogel et al ldquoNovel small RNA-encoding genes in the intergenic regions of Escherichia colirdquoCurrent Biology vol 11 no 12 pp 941ndash950 2001

[8] Y Chao K Papenfort R Reinhardt CM Sharma and J VogelldquoAn atlas ofHfq-bound transcripts reveals 31015840 UTRs as a genomicreservoir of regulatory small RNAsrdquoThe EMBO Journal vol 31no 20 pp 4005ndash4019 2012

[9] G Klein N Kobylak B Lindner A Stupak and S RainaldquoAssembly of lipopolysaccharide in Escherichia coli requiresthe essential LapB heat shock proteinrdquo Journal of BiologicalChemistry vol 289 no 21 pp 14829ndash14853 2014

[10] J Vogel and B F Luisi ldquoHfq and its constellation of RNArdquoNature Reviews Microbiology vol 9 no 8 pp 578ndash589 2011

[11] P Babitzke and T Romeo ldquoCsrB sRNA family sequestration ofRNA-binding regulatory proteinsrdquo Current Opinion in Microbi-ology vol 10 no 2 pp 156ndash163 2007

[12] K M Wassarman ldquo6S RNA a small RNA regulator of tran-scriptionrdquo Current Opinion in Microbiology vol 10 no 2 pp164ndash168 2007

[13] C Dartigalongue DMissiakas and S Raina ldquoCharacterizationof the Escherichia coli 120590E regulonrdquo The Journal of BiologicalChemistry vol 276 no 24 pp 20866ndash20875 2001

[14] S Raina D Missiakas and C Georgopoulos ldquoThe rpoE geneencoding the 120590E (12059024) heat shock sigma factor of EscherichiacolirdquoThe EMBO Journal vol 14 no 5 pp 1043ndash1055 1995

[15] P E Rouviere A De Las Penas J Mecsas C Z Lu K E Ruddand C A Gross ldquorpoE the gene encoding the second heat-shock sigma factor 120590E in Escherichia colirdquoThe EMBO Journalvol 14 no 5 pp 1032ndash1042 1995

[16] D Missiakas M P Mayer M Lemaire C Georgopoulos andS Raina ldquoModulation of the Escherichia coli120590119864 (RpoE) heat-shock transcription-factor activity by the RseA RseB and RseCproteinsrdquo Molecular Microbiology vol 24 no 2 pp 355ndash3711997

[17] A A Rasmussen M Eriksen K Gilany et al ldquoRegulation ofompA mRNA stability the role of a small regulatory RNA ingrowth phase-dependent controlrdquoMolecular Microbiology vol58 no 5 pp 1421ndash1429 2005

[18] J Johansen A A Rasmussen M Overgaard and P Valentin-Hansen ldquoConserved small non-coding RNAs that belong tothe 120590E regulon role in down-regulation of outer membraneproteinsrdquo Journal of Molecular Biology vol 364 no 1 pp 1ndash82006

[19] K Papenfort V Pfeiffer F Mika S Lucchini J C D Hintonand J Vogel ldquo120590E-dependent small RNAs of Salmonella respondto membrane stress by accelerating global omp mRNA decayrdquoMolecular Microbiology vol 62 no 6 pp 1674ndash1688 2006

[20] K I Udekwu and E GHWagner ldquoSigma E controls biogenesisof the antisense RNAMicArdquoNucleic Acids Research vol 35 no4 pp 1279ndash1288 2007

[21] A Coornaert A Lu P Mandin M Springer S Gottesmanand M Guillier ldquoMicA sRNA links the PhoP regulon to cellenvelope stressrdquoMolecular Microbiology vol 76 no 2 pp 467ndash479 2010

[22] M S Guo T B Updegrove E B Gogol S A Shabalina C AGross andG Storz ldquoMicL a new120590E-dependent sRNA combatsenvelope stress by repressing synthesis of Lpp the major outermembrane lipoproteinrdquo Genes amp Development vol 28 no 14pp 1620ndash1634 2014


[23] R Noor M Murata H Nagamitsu G Klein S Raina and MYamada ldquoDissection of 120590E-dependent cell lysis in Escherichiacoli roles of RpoE regulators RseA RseB and periplasmicfolding catalyst PpiDrdquo Genes to Cells vol 14 no 7 pp 885ndash8992009

[24] E B Gogol V A Rhodius K Papenfort J Vogel and CA Gross ldquoSmall RNAs endow a transcriptional activator withessential repressor functions for single-tier control of a globalstress regulonrdquo Proceedings of the National Academy of Sciencesof the United States of America vol 108 no 31 pp 12875ndash128802011

[25] G Klein B Lindner H Brade and S Raina ldquoMolecularbasis of lipopolysaccharide heterogeneity in Escherichia colienvelope stress-responsive regulators control the incorporationof glycoforms with a third 3-deoxy-120572-D-manno-oct-2-ulosonicacid and rhamnoserdquo The Journal of Biological Chemistry vol286 no 50 pp 42787ndash42807 2011

[26] H Nikaido ldquoOuter membranerdquo in Escherichia coli andSalmonella Cellular and Molecular Biology F C NeidhardtR Curtiss J L Ingraham et al Eds American Society forMicrobiology Press Washington DC USA 1996

[27] R R Kulesus K Diaz-Perez E S Slechta D S Eto and M AMulvey ldquoImpact of the RNA chaperone Hfq on the fitness andvirulence potential of uropathogenic Escherichia colirdquo Infectionand Immunity vol 76 no 7 pp 3019ndash3026 2008

[28] G Klein S Muller-Loennies B Lindner N Kobylak H Bradeand S Raina ldquoMolecular and structural basis of inner corelipopolysaccharide alterations in Escherichia coli incorporationof glucuronic acid and phosphoethanolamine in the heptoseregionrdquoThe Journal of Biological Chemistry vol 288 no 12 pp8111ndash8127 2013

[29] S Muller-Loennies B Lindner and H Brade ldquoStructuralanalysis of oligosaccharides from lipopolysaccharide (LPS) ofEscherichia coli K12 strain W3100 reveals a link between innerand outer core LPS biosynthesisrdquo The Journal of BiologicalChemistry vol 278 no 36 pp 34090ndash34101 2003

[30] C R H Raetz and C Whitfield ldquoLipopolysaccharide endotox-insrdquo Annual Review of Biochemistry vol 71 pp 635ndash700 2002

[31] K Young L L SilverD Bramhill et al ldquoThe envApermeabilitycell division gene of Escherichia coli encodes the second enzymeof lipid A biosynthesis UDP-3-O-(R-3-hydroxymyristoyl)-N-acetylglucosamine deacetylaserdquoThe Journal of Biological Chem-istry vol 270 no 51 pp 30384ndash30391 1995

[32] T C Meredith U Mamat Z Kaczynski B Lindner O Holstand R W Woodard ldquoModification of lipopolysaccharide withcolanic acid (M-antigen) repeats inEscherichia colirdquoThe Journalof Biological Chemistry vol 282 no 11 pp 7790ndash7798 2007

[33] E Pinta Z Li J Batzilla et al ldquoIdentification of threeoligo-polysaccharide-specific ligases in Yersinia enterocoliticardquoMolecular Microbiology vol 83 no 1 pp 125ndash136 2012

[34] J Plumbridge andEVimr ldquoConvergent pathways for utilizationof the amino sugarsN-acetylglucosamineN-acetylmannosam-ine andN-acetylneuraminic acid by Escherichia colirdquo Journal ofBacteriology vol 181 no 1 pp 47ndash54 1999

[35] F Kalamorz B Reichenbach W Marz B Rak and B GorkeldquoFeedback control of glucosamine-6-phosphate synthase GlmSexpression depends on the small RNA GlmZ and involves thenovel protein YhbJ in Escherichia colirdquoMolecular Microbiologyvol 65 no 6 pp 1518ndash1533 2007

[36] Y Gopel K Papenfort B Reichenbach J Vogel and B GorkeldquoTargeted decay of a regulatory small RNA by an adaptor

protein for RNase E and counteraction by an anti-adaptorRNArdquo Genes amp Development vol 27 no 5 pp 552ndash564 2013

[37] K S Frohlich and J Vogel ldquoActivation of gene expression bysmall RNArdquo Current Opinion in Microbiology vol 12 no 6 pp674ndash682 2009

[38] Y Gopel M A Khan and B Gorke ldquoMenage a trois post-transcriptional control of the key enzyme for cell envelopesynthesis by a base-pairing small RNA an RNase adaptorprotein and a small RNA mimicrdquo RNA Biology vol 11 no 5pp 433ndash442 2014

[39] J H Urban and J Vogel ldquoTwo seemingly homologous noncod-ing RNAs act hierarchically to activate glmSmRNA translationrdquoPLoS Biology vol 6 no 3 article e64 2008

[40] B Reichenbach A Maes F Kalamorz E Hajnsdorf and BGorke ldquoThe small RNAGlmY acts upstreamof the sRNAGlmZin the activation of glmS expression and is subject to regulationby polyadenylation in Escherichia colirdquo Nucleic Acids Researchvol 36 no 8 pp 2570ndash2580 2008

[41] P Sperandeo R Cescutti R Villa et al ldquoCharacterization oflptA and lptB two essential genes implicated in lipopolysac-charide transport to the outer membrane of Escherichia colirdquoJournal of Bacteriology vol 189 no 1 pp 244ndash253 2007

[42] J Njoroge and V Sperandio ldquoEnterohemorrhagic Escherichiacoli virulence regulation by two bacterial adrenergic kinasesQseC andQseErdquo Infection and Immunity vol 80 no 2 pp 688ndash703 2012

[43] M S Anderson C E Bulawa and C R H Raetz ldquoThebiosynthesis of gram-negative endotoxin Formation of lipid Aprecursors from UDP-GlcNAc in extracts of Escherichia colirdquoThe Journal of Biological Chemistry vol 260 no 29 pp 15536ndash15541 1985

[44] T M Kelly S A Stachula C R H Raetz and M S AndersonldquoThe firA gene of Escherichia coli encodes UDP-3-O-(R-3-hydroxymyristoyl)-glucosamine N-acyltransferase The thirdstep of endotoxin biosynthesisrdquoThe Journal of Biological Chem-istry vol 268 no 26 pp 19866ndash19874 1993

[45] S Mohan T M Kelly S S Eveland C R H Raetz and MS Anderson ldquoAn Escherichia coli gene (FabZ) encoding (3R)-hydroxymyristoyl acyl carrier protein dehydrase Relation tofabA and suppression of mutations in lipid A biosynthesisrdquoTheJournal of Biological Chemistry vol 269 no 52 pp 32896ndash32903 1994

[46] T Ogura K Inoue T Tatsuta et al ldquoBalanced biosynthesis ofmajor membrane components through regulated degradationof the committed enzyme of lipid A biosynthesis by the AAAprotease FtsH (HflB) in Escherichia colirdquo Molecular Microbiol-ogy vol 31 no 3 pp 833ndash844 1999

[47] F Fuhrer S Langklotz and F Narberhaus ldquoThe C-terminalend of LpxC is required for degradation by the FtsH proteaserdquoMolecular Microbiology vol 59 no 3 pp 1025ndash1036 2006

[48] S Langklotz M Schakermann and F Narberhaus ldquoControl oflipopolysaccharide biosynthesis by FtsH-mediated proteolysisof LpxC is conserved in enterobacteria but not in all gram-negative bacteriardquo Journal of Bacteriology vol 193 no 5 pp1090ndash1097 2011

[49] E Sonnleitner T Sorger-Domenigg M J Madej et al ldquoDetec-tion of small RNAs inPseudomonas aeruginosa byRNomics andstructure-based bioinformatic toolsrdquoMicrobiology vol 154 no10 pp 3175ndash3187 2008

[50] E Sonnleitner and D Haas ldquoSmall RNAs as regulators ofprimary and secondary metabolism in Pseudomonas speciesrdquo


Applied Microbiology and Biotechnology vol 91 no 1 pp 63ndash79 2011

[51] A P Tomaras C JMcPhersonMKuhn et al ldquoLpxC Inhibitorsas new antibacterial agents and tools for studying regulation oflipid a biosynthesis in Gram-negative Pathogensrdquo mBio vol 5no 5 Article ID e01551 2014

[52] R E Caughlan A K Jones A M DeLucia et al ldquoMechanismsdecreasing in vitro susceptibility to the LpxC inhibitor CHIR-090 in the gram-negative pathogen Pseudomonas aeruginosardquoAntimicrobial Agents and Chemotherapy vol 56 no 1 pp 17ndash27 2012

[53] S M Carty K R Sreekumar and C R H Raetz ldquoEffect of coldshock on lipid a biosynthesis in Escherichia coli Induction at12∘C of an acyltransferase specific for palmitoleoyl-acyl carrierproteinrdquoThe Journal of Biological Chemistry vol 274 no 14 pp9677ndash9685 1999

[54] G Klein B Lindner W Brabetz H Brade and S RainaldquoEscherichia coli K-12 suppressor-free mutants lacking earlyglycosyltransferases and late acyltransferases minimallipopolysaccharide structure and induction of envelope stressresponserdquo The Journal of Biological Chemistry vol 284 no 23pp 15369ndash15389 2009

[55] S Lacour E Bechet A J Cozzone I Mijakovic and CGrangeasse ldquoTyrosine phosphorylation of the UDP-glucosedehydrogenase of Escherichia coli is at the crossroads of colanicacid synthesis and polymyxin resistancerdquo PLoS ONE vol 3 no8 Article ID e3053 2008

[56] K Moon and S Gottesman ldquoA PhoQP-regulated small RNAregulates sensitivity of Escherichia coli to antimicrobial pep-tidesrdquo Molecular Microbiology vol 74 no 6 pp 1314ndash13302009

[57] K Moon D A Six H-J Lee C R H Raetz and S GottesmanldquoComplex transcriptional and post-transcriptional regulationof an enzyme for lipopolysaccharide modificationrdquo MolecularMicrobiology vol 89 no 1 pp 52ndash64 2013

[58] Z Zhou S Lin R J Cotter and C R Raetz ldquoLipid Amodifica-tions characteristic of Salmonella typhimurium are induced byNH4VO3inEscherichia coliK12 detection of 4-amino-4-deoxy-

l-arabinose phosphoethanolamine and palmitaterdquo The Journalof Biological Chemistry vol 274 no 26 pp 18503ndash18514 1999

[59] C Tam and D Missiakas ldquoChanges in lipopolysaccharidestructure induce the 120590E-dependent response of Escherichia colirdquoMolecular Microbiology vol 55 no 5 pp 1403ndash1412 2005

[60] C R H Raetz C M Reynolds M S Trent and R E BishopldquoLipid A modification systems in gram-negative bacteriardquoAnnual Review of Biochemistry vol 76 pp 295ndash329 2007

[61] B D Needham and M S Trent ldquoFortifying the barrier theimpact of lipidA remodelling on bacterial pathogenesisrdquoNatureReviews Microbiology vol 11 no 7 pp 467ndash481 2013

[62] A Kato T Latifi and E A Groisman ldquoClosing the loopthe PmrAPmrB two-component system negatively controlsexpression of its posttranscriptional activator PmrDrdquo Proceed-ings of the National Academy of Sciences of the United States ofAmerica vol 100 no 8 pp 4706ndash4711 2003

[63] E J Rubin C M Herrera A A Crofts andM S Trent ldquoPmrDis required for modifications to Escherichia coli endotoxin thatpromote antimicrobial resistancerdquo Antimicrobial Agents andChemotherapy vol 59 no 4 pp 2051ndash2061 2015

[64] H Ogasawara S Shinohara K Yamamoto and A IshihamaldquoNovel regulation targets of themetal-response BasS-BasR two-component system of Escherichia colirdquo Microbiology vol 158no 6 pp 1482ndash1492 2012

[65] S C Pulvermacher L T Stauffer and G V Stauffer ldquoRoleof the sRNA GcvB in regulation of cycA in Escherichia colirdquoMicrobiology vol 155 no 1 pp 106ndash114 2009

[66] C M Sharma K Papenfort S R Pernitzsch H-J MollenkopfJ C D Hinton and J Vogel ldquoPervasive post-transcriptionalcontrol of genes involved in amino acidmetabolism by the Hfq-dependent GcvB small RNArdquo Molecular Microbiology vol 81no 5 pp 1144ndash1165 2011

[67] M Miyakoshi Y Chao and J Vogel ldquoCross talk between ABCtransporter mRNAs via a target mRNA-derived sponge of theGcvB small RNArdquoThe EMBO Journal vol 34 no 11 pp 1478ndash1492 2015

[68] A Coornaert C Chiaruttini M Springer and M GuillierldquoPost-transcriptional control of the Escherichia coli PhoQ-PhoPtwo-component system by multiple sRNAs involves a novelpairing region of GcvBrdquo PLoS Genetics vol 9 no 1 Article IDe1003156 2013

[69] C P Corcoran D Podkaminski K Papenfort J H Urban JC D Hinton and J Vogel ldquoSuperfolder GFP reporters validatediverse new mRNA targets of the classic porin regulator MicFRNArdquoMolecular Microbiology vol 84 no 3 pp 428ndash445 2012

[70] S Amini H Goodarzi and S Tavazoie ldquoGenetic dissectionof an exogenously induced biofilm in laboratory and clinicalisolates of E colirdquo PLoS Pathogens vol 5 no 5 Article IDe1000432 2009

[71] S P Pandey J A Winkler H Li D M Camacho J JCollins and G C Walker ldquoCentral role for RNase YbeY inHfq-dependent and Hfq-independent small-RNA regulation inbacteriardquo BMC Genomics vol 15 article 121 2014

[72] C M Reynolds S R Kalb R J Cotter and C R H RaetzldquoA phosphoethanolamine transferase specific for the outer 3-deoxy-D-manno-octulosonic acid residue of Escherichia colilipopolysaccharide Identification of the eptB gene and Ca2+hypersensitivity of an eptB deletion mutantrdquo The Journal ofBiological Chemistry vol 280 no 22 pp 21202ndash21211 2005

[73] A Kato H D Chen T Latifi and E A Groisman ldquoReciprocalcontrol between a bacteriumrsquos regulatory system and the modi-fication status of its lipopolysacchariderdquoMolecular Cell vol 47no 6 pp 897ndash908 2012

[74] C M Reynolds A A Ribeiro S C McGrath R J Cotter CR H Raetz and M S Trent ldquoAn outer membrane enzymeencoded by Salmonella typhimurium lpxR that removes the31015840acyloxyacyl moiety of lipid Ardquo The Journal of BiologicalChemistry vol 281 no 31 pp 21974ndash21987 2006

[75] M Reines E Llobet K M Dahlstrom et al ldquoDecipheringthe acylation pattern of Yersinia enterocolitica lipid Ardquo PLoSPathogens vol 8 no 10 Article ID e1002978 2012

[76] EGuisbert VA RhodiusNAhuja EWitkin andCAGrossldquoHfq modulates the 120590E-mediated envelope stress response andthe 12059032-mediated cytoplasmic stress response in Escherichiacolirdquo Journal of Bacteriology vol 189 no 5 pp 1963ndash1973 2007

[77] C Yang T-W Huang S-Y Wen et al ldquoGenome-wide PhoBbinding and gene expression profiles reveal the hierarchicalgene regulatory network of phosphate starvation in Escherichiacolirdquo PLoS ONE vol 7 no 10 Article ID e47314 2012

[78] J Klena P Zhang O Schwartz S Hull and T Chen ldquoThecore lipopolysaccharide of Escherichia coli is a ligand for thedendritic-cell-specific intercellular adhesionmolecule noninte-grin CD209 receptorrdquo Journal of Bacteriology vol 187 no 5 pp1710ndash1715 2005

[79] H S Gibbons S R Kalb R J Cotter and C R H RaetzldquoRole of Mg2+ and pH in the modification of Salmonella lipid


A after endocytosis by macrophage tumour cellsrdquo MolecularMicrobiology vol 55 no 2 pp 425ndash440 2005

[80] L Guo K B Lim C M Poduje et al ldquoLipid A acylation andbacterial resistance against vertebrate antimicrobial peptidesrdquoCell vol 95 no 2 pp 189ndash198 1998

[81] S D Breazeale A A Ribeiro A L McClerren and C R HRaetz ldquoA formyltransferase required for polymyxin resistanceinEscherichia coli and themodification of lipidAwith 4-amino-4-deoxy-L-arabinose Identification and function of UDP-4-deoxy-4-formamido-L-arabinoserdquo The Journal of BiologicalChemistry vol 280 no 14 pp 14154ndash14167 2005

[82] R Tamayo B Choudhury A Septer M Merighi R Carlsonand J S Gunn ldquoIdentification of cptA a PmrA-regulatedlocus required for phosphoethanolamine modification of theSalmonella enterica serovar Typhimurium lipopolysaccharidecorerdquo Journal of Bacteriology vol 187 no 10 pp 3391ndash33992005

[83] S C Earl M T Rogers J Keen et al ldquoResistance to innateimmunity contributes to colonization of the insect gut byYersinia pestisrdquo PLoS ONE vol 10 no 7 Article ID e01333182015

[84] C Ansong H Yoon S Porwollik et al ldquoGlobal systems-levelanalysis of Hfq and SmpB deletion mutants in Salmonellaimplications for virulence and global protein translationrdquo PLoSONE vol 4 no 3 Article ID e4809 2009

[85] S Palaniyandi A Mitra C D Herren et al ldquoBarA-UvrY two-component system regulates virulence of uropathogenic E coliCFT073rdquo PLoS ONE vol 7 no 2 Article ID e31348 2012

[86] T Romeo M Gong Mu Ya Liu and A-M Brun-ZinkernagelldquoIdentification and molecular characterization of csrA apleiotropic gene from Escherichia coli that affects glycogenbiosynthesis gluconeogenesis cell size and surface propertiesrdquoJournal of Bacteriology vol 175 no 15 pp 4744ndash4755 1993

[87] T Romeo C A Vakulskas and P Babitzke ldquoPost-trans-criptional regulation on a global scale form and function ofCsrRsm systemsrdquo Environmental Microbiology vol 15 no 2pp 313ndash324 2013

[88] A L Gore and S M Payne ldquoCsrA and Cra influence Shigellaflexneri pathogenesisrdquo Infection and Immunity vol 78 no 11pp 4674ndash4682 2010

[89] B W Bainbridge T Hirano N Grieshaber M E Davey andG A OrsquoToole ldquoDeletion of a 77-base-pair inverted repeatelement alters the synthesis of surface polysaccharides inPorphyromonas gingivalisrdquo Journal of Bacteriology vol 197 no7 pp 1208ndash1220 2015

[90] T Moslashller T Franch C Udesen K Gerdes and P Valentin-Hansen ldquoSpot 42RNAmediates discoordinate expression of theE coli galactose operonrdquo Genes amp Development vol 16 no 13pp 1696ndash1706 2002

[91] A E Smith S-H Kim F Liu et al ldquoPagP activation in the outermembrane triggers R3 core oligosaccharide truncation in thecytoplasm of Escherichia coliO157H7rdquoThe Journal of BiologicalChemistry vol 283 no 7 pp 4332ndash4343 2008


examples of such RNAs include E coli CsrA binding CsrB-CsrC sRNAs and 6S RNA which binds to housekeeping formof RNA polymerase E12059070 [11 12] Tight regulation mediatedby sRNAs is often the result of interconnected circuits actingin feedforward or feedback mechanisms that can regulatetranscriptional factors and two-component systems consti-tuting nodal controls of complex biological networks HenceGram-negative bacteria like Escherichia coli encode over 100sRNAs and several of them have been well characterizedregulating diverse cellular pathways [1ndash3]

In order to achieve high sensitivity many sRNAs aretranscribed in response to specific signals The transcriptionof such sRNAs is often regulated by specialized sigma factorsor by two-component systems In turn sRNAs may providethe feed-back mechanism for the downregulation of sigmafactors and two-component systems once the signal is dealtwith Often downregulation by sRNAs is achieved by reduc-ing the synthesis or translation of these regulatorsThis allowsfor the dampening of elevated stress responses or alteredmetabolic pathways

One of the most well characterized stress responsivesigma factor is RpoE which controls the transcription ofseveral genes with a dedicated function in the envelopebiogenesis (Figure 1) Thus RpoE regulon members in Ecoli include genes encoding periplasmic folding factors (surAfkpA skp and dsbC) proteases [degP ecfE (rseP)] compo-nents of outer membrane proteins (OMPs) assembly (bamA-E) genes whose products are involved in lipopolysaccharide(LPS) translocation and assembly (lptA lptB and lptD)synthesis of phospholipids and lipid A (lpxP lpxD fabZlpxA and lpxB) and the rpoH gene encoding heat shocksigma factor [13] RpoE positively autoregulates its owntranscription and regulates its own activity negatively bytranscribing genes (rseA and rseB) whose products act asnegative regulators [14ndash16] RpoE regulon also includes threesRNAs (RybB MicA and SlrA) [9 17ndash22]

Induction of RpoEupon envelope stress or due to deletionof antisigma factor RseA leads to pronounced reduction inthe amounts ofOMPs Lpp lipoprotein and alterations in LPScomposition [9 22ndash25] The repression of OMPs synthesisoccurs at the posttranscriptional level wherein base-paringsRNAs like MicA inhibits translation and decay of the ompAmRNA and RybB inhibits translation of ompC ompW andompN mRNAs [17ndash19] This inhibition in the synthesis ofmajor OMPs under stress conditions is to maintain an enve-lope homeostasis as some of these OMPs constitute majorabundant proteins under normal growth conditions ThusRpoE-regulated sRNAs MicA RybB and SlrA provide oneof the best examples of the positive regulation (feedforwardmechanism) byRpoE in response to envelope stress andRpoEdownregulation by these sRNAs in a feedback mechanism(Figure 1) [9 24] The orchestrated activities of RpoE MicARybB and SlrA insure homeostatic control of outer mem-brane components such as LPS most abundant lipoproteinLpp and OMPs Such a regulation also allows integration ofdiverse signals from two-component systems with the RpoEsigma factorThere exists an in-built link between OMP con-tent and LPS structure Severe defects in LPS trigger massive

induction of RpoE and this can simply explain reduction inthe amounts of OMPs due to their downregulation of synthe-sis by MicA- and RybB-mediated translational repression

The outer membrane of Gram-negative bacteria is com-posed of highly abundant OMPs and LPS Approximately 2 times106 molecules of LPS cover nearly 75 of the cell surface[26]Thus cellular regulatory controls are in place tomonitorthe biogenesis of OMPs and LPS In many Gram-negativebacteria sRNAs have been shown to play an important rolefor the homeostasis of the envelope In this review specificsRNAs encoded in the E coli genome that regulate thesynthesis composition and structural modification of LPSan essential component of the cell envelope are highlightedAs will be evident specific sRNAs control events in LPSbiosynthesis from its early steps regulating a balance betweenamounts of fatty acids (phospholipids) and LPS as well asincorporation of specific nonstoichiometric modifications inLPS These structural alterations are critical for impartingresistance to antibiotics and survival under defined envi-ronmental niches (Table 1) Dedicated sRNAs most notablycontrol the accumulation of specific LPS glycoforms therebycontrolling LPS composition and its heterogeneity (Figures 12 and 5) The ability to generate glycoforms with truncationsin the outer core of LPS ismodulated by sRNAs andRpoE andis critical for the addition of O-antigen that confers serumresistance [25] In pathogenic Gram-negative bacteria LPSis known to be a major virulence factor Consistent withthe key role of LPS in bacterial virulence the attenuatedvirulence phenotype of hfq mutants can be ascribed toLPS alterations that are regulated by Hfq-dependent sRNAs[1 27]

2 LPS Biosynthesis and Its Heterogeneity

The cytoplasm of Gram-negative bacteria such as E coli issurrounded by an inner membrane (IM) that separates anaqueous periplasmic compartment containing peptidoglycanfrom the outer membrane (OM) The OM is asymmetric innature with phospholipids facing the inner leaflet and theLPS facing towards the outside LPS is a complex glycolipidand constitutes themajor amphiphilic component of theOMIts chemical composition contributes to the permeabilitybarrier function The synthesis of LPS and phospholipidsis tightly coregulated and held at a nearly constant ratio of015 to 10 [26] However LPS is highly heterogeneous incomposition and comprised of a mixture of different glyco-forms [25 28 29] The ratio of different glycoforms whichdiffer due to nonstoichiometric substitutions is regulatedby various stress-responsive two-component systems such asPhoBR (sensing phosphate concentration) PhoPQ (sensingdivalent cations like Mg2+) and BasSR (responsive to Fe3+Zn2+ antimicrobial peptides) and above all by the sigmafactor RpoE the master regulator of envelope biogenesisprocesses [25] Embedded within these regulatory systemsare several noncoding sRNAs (MicA RybB SlrA MgrR andArcZ) that in turn regulate the incorporation of some of thesenonstoichiometric modifications and hence control the LPSheterogeneity (Figures 1 and 2)


RybB

RseA

LptBLptD


FtsH

LpxCWaaA


WaaAWaaCWaaF

WaaOWaaB

WaaG

RfaH

WaaR

FabZPhoPQ

PhoBR

WaaHUgd

BasSR

PmrR

LpxT

EptC

Rcs RprA RpoS

LpxPC161


WaaR

MgrREptB

WaaZ

ppGpp

PagPC160

Defective LPS


ArcZGcvB


Lpp

PG


GalK

Spot42

LPSLapB

assembly

GadY

MicA

CsrB

MicA

Coremodifications


HO

HO OHO

O OOH

HOO

O OHOH

HOHO

HO

HO

HOO

O

O

OOOO

O OOH

HO

HO

OOH

O

OO

OHOHP

P

NHNH

141414

14

HO

HO

HO

HOO

O

O

OOOO

O OO

HO

HO

OOH

O

OO

OHOHP

P

NHNH

141414

14

HO

120590E

12059032

l-Ara4N

l-Ara4N











[25]



[70]










galK

Spot42

CRP

cAMP

Glucose uptake

SgrS

LPS

prec

urso

rs

Export of LPSto OM

Deacylation

Acylation with C16

LpxR

MicF

lpxR

pagP

PhoP

MicA

PagP


pmrR

BasSR

PmrR

LpxT

lpxT


O

OH

HOHO

OONH

UDP

LpxA

14

OOH

HO

O ONH UDP

O

HO

O

LpxC

FabZ O

ACP

FabIPhospholipids

PE

PG

UDP-GlcNAc

SlrA Lpp

Core biosynthesis

P-EtN onthe second

Kdo

eptBArcZ

MicAPhoPQ

P-EtN

GcvBMgrR

lpxT waaRGlcIII



GlmZY

waaZpmrD

BasSRglmS

waaB waaS

ACP

OH

ACP

14

OOH

HO

O UDP

O

HO

O

UDP-GlcUDP-Gal


arnTeptA


LPS translocation

CH3-(CH2)10

CH3-(CH2)10 CH3-(CH2)10

NH2

Kdo2-lipid A

R-3-OH-C14-ACP


O


l-Ara4N

O







AdaptorRapZ

AdaptorRapZ

AdaptorRapZ

AdaptorRapZ



SD

GlmS

GlcN6P UDP-GlcNAc


Peptidoglycan



Translation

Hfq

glmS mRNA

Complex

Processing

GlmZ

GlmY


glmS mRNA

RNase E

ProcessingglmS mRNA

SD Translation

Degradation

Complex

Processing

RNase E

UGA

GlmZ GlmZ

GlmY

GlmZ

GlmY

GlmZ GlmZ

GlmY

Free

Transcription

LowGlcN6P

HighGlcN6P

(a)

(b)

(c)

RapZ

RapZ




















2-lipid A) and











HO

HO

HO

HO

HO

OH

O

O

O

O

OO

OO

O

O

OH

HOO

O

O

O O

O

OH

O

OHO

OH

O

OO

OH

OH

OH

HO

P

P

NH

NH

12

1414

1414

HO

O

14

O

Lipid A

Kdo2

(a)

O

14

HO

HO

HO

OH

O

O

O

O

OO

O O

O

O

OH

HOO

O

OO O

O

OH

O

OO

OHO

O

OO

OOH

R2

OHHO

P

P

NH

NH

12

14

14 1414

HO

HOO

OH

O

R1 O

O

OHP

EptA

OO

OP P

LpxT

RdlA

MicA

OO

OH

EptBMgrR

LpxRMicF

ArcZ

O

16PagP

O

OHOHOH

NH2

NH2

PNH2

(b)









Lipid A

2

4

WaaAWaaQ7

1

PWaaP

44

PWaaY

5 KdoI 2

KdoII

WaaA

HepIII


WaaG

6

1WaaU

HepIV

GlcIII 1

WaaR

WaaB6

1

Gal

Glycoform I

WaaCGlcII 12

WaaO WaaF

(a)

Lipid A

2

4

PEptC

P-EtN

1

6

WaaSRha

2

4

WaaZ

Glycoform IV


Gal

HepI 1 53 KdoI 2

KdoII 5

1

7

44

P

MgrR

ArcZEptB

RybBWaaRRpoE

KdoIII

HepIII 1

RpoE

Hfq

MicAlowast

(b)

Lipid A

2

4

PEptC

P-EtN

1

6

EptB

2

4

WaaZ

Glycoform V


Gal

HepI 1 53 KdoI 2

KdoII 7

1

7

1

WaaSRha5

P-EtN

44

P

KdoIII

HepIII

RybBWaaRRpoE

RpoE

Hfq

RpoE

MicAlowast

(c)

Lipid A

2

4

PEptC

P-EtN

1

6

EptB

2

4

WaaZ


Gal HepIII

HepI 13 5 KdoI 2

KdoII 7

1

7

1

WaaSRha5

P-EtN

4

WaaH7

1

GlcUA

Glycoform VII

KdoIII

RybBWaaRRpoE

RpoE

HfqRpoE

MicAlowast

(d)


























11 Conclusion






Acknowledgments


References




































































































RybB

RseA

LptBLptD


FtsH

LpxCWaaA


WaaAWaaCWaaF

WaaOWaaB

WaaG

RfaH

WaaR

FabZPhoPQ

PhoBR

WaaHUgd

BasSR

PmrR

LpxT

EptC

Rcs RprA RpoS

LpxPC161


WaaR

MgrREptB

WaaZ

ppGpp

PagPC160

Defective LPS


ArcZGcvB


Lpp

PG


GalK

Spot42

LPSLapB

assembly

GadY

MicA

CsrB

MicA

Coremodifications


HO

HO OHO

O OOH

HOO

O OHOH

HOHO

HO

HO

HOO

O

O

OOOO

O OOH

HO

HO

OOH

O

OO

OHOHP

P

NHNH

141414

14

HO

HO

HO

HOO

O

O

OOOO

O OO

HO

HO

OOH

O

OO

OHOHP

P

NHNH

141414

14

HO

120590E

12059032

l-Ara4N

l-Ara4N











[25]



[70]










galK

Spot42

CRP

cAMP

Glucose uptake

SgrS

LPS

prec

urso

rs

Export of LPSto OM

Deacylation

Acylation with C16

LpxR

MicF

lpxR

pagP

PhoP

MicA

PagP


pmrR

BasSR

PmrR

LpxT

lpxT


O

OH

HOHO

OONH

UDP

LpxA

14

OOH

HO

O ONH UDP

O

HO

O

LpxC

FabZ O

ACP

FabIPhospholipids

PE

PG

UDP-GlcNAc

SlrA Lpp

Core biosynthesis

P-EtN onthe second

Kdo

eptBArcZ

MicAPhoPQ

P-EtN

GcvBMgrR

lpxT waaRGlcIII



GlmZY

waaZpmrD

BasSRglmS

waaB waaS

ACP

OH

ACP

14

OOH

HO

O UDP

O

HO

O

UDP-GlcUDP-Gal


arnTeptA


LPS translocation

CH3-(CH2)10

CH3-(CH2)10 CH3-(CH2)10

NH2

Kdo2-lipid A

R-3-OH-C14-ACP


O


l-Ara4N

O







AdaptorRapZ

AdaptorRapZ

AdaptorRapZ

AdaptorRapZ



SD

GlmS

GlcN6P UDP-GlcNAc


Peptidoglycan



Translation

Hfq

glmS mRNA

Complex

Processing

GlmZ

GlmY


glmS mRNA

RNase E

ProcessingglmS mRNA

SD Translation

Degradation

Complex

Processing

RNase E

UGA

GlmZ GlmZ

GlmY

GlmZ

GlmY

GlmZ GlmZ

GlmY

Free

Transcription

LowGlcN6P

HighGlcN6P

(a)

(b)

(c)

RapZ

RapZ




















2-lipid A) and











HO

HO

HO

HO

HO

OH

O

O

O

O

OO

OO

O

O

OH

HOO

O

O

O O

O

OH

O

OHO

OH

O

OO

OH

OH

OH

HO

P

P

NH

NH

12

1414

1414

HO

O

14

O

Lipid A

Kdo2

(a)

O

14

HO

HO

HO

OH

O

O

O

O

OO

O O

O

O

OH

HOO

O

OO O

O

OH

O

OO

OHO

O

OO

OOH

R2

OHHO

P

P

NH

NH

12

14

14 1414

HO

HOO

OH

O

R1 O

O

OHP

EptA

OO

OP P

LpxT

RdlA

MicA

OO

OH

EptBMgrR

LpxRMicF

ArcZ

O

16PagP

O

OHOHOH

NH2

NH2

PNH2

(b)









Lipid A

2

4

WaaAWaaQ7

1

PWaaP

44

PWaaY

5 KdoI 2

KdoII

WaaA

HepIII


WaaG

6

1WaaU

HepIV

GlcIII 1

WaaR

WaaB6

1

Gal

Glycoform I

WaaCGlcII 12

WaaO WaaF

(a)

Lipid A

2

4

PEptC

P-EtN

1

6

WaaSRha

2

4

WaaZ

Glycoform IV


Gal

HepI 1 53 KdoI 2

KdoII 5

1

7

44

P

MgrR

ArcZEptB

RybBWaaRRpoE

KdoIII

HepIII 1

RpoE

Hfq

MicAlowast

(b)

Lipid A

2

4

PEptC

P-EtN

1

6

EptB

2

4

WaaZ

Glycoform V


Gal

HepI 1 53 KdoI 2

KdoII 7

1

7

1

WaaSRha5

P-EtN

44

P

KdoIII

HepIII

RybBWaaRRpoE

RpoE

Hfq

RpoE

MicAlowast

(c)

Lipid A

2

4

PEptC

P-EtN

1

6

EptB

2

4

WaaZ


Gal HepIII

HepI 13 5 KdoI 2

KdoII 7

1

7

1

WaaSRha5

P-EtN

4

WaaH7

1

GlcUA

Glycoform VII

KdoIII

RybBWaaRRpoE

RpoE

HfqRpoE

MicAlowast

(d)


























11 Conclusion






Acknowledgments


References










































































































[25]



[70]










galK

Spot42

CRP

cAMP

Glucose uptake

SgrS

LPS

prec

urso

rs

Export of LPSto OM

Deacylation

Acylation with C16

LpxR

MicF

lpxR

pagP

PhoP

MicA

PagP


pmrR

BasSR

PmrR

LpxT

lpxT


O

OH

HOHO

OONH

UDP

LpxA

14

OOH

HO

O ONH UDP

O

HO

O

LpxC

FabZ O

ACP

FabIPhospholipids

PE

PG

UDP-GlcNAc

SlrA Lpp

Core biosynthesis

P-EtN onthe second

Kdo

eptBArcZ

MicAPhoPQ

P-EtN

GcvBMgrR

lpxT waaRGlcIII



GlmZY

waaZpmrD

BasSRglmS

waaB waaS

ACP

OH

ACP

14

OOH

HO

O UDP

O

HO

O

UDP-GlcUDP-Gal


arnTeptA


LPS translocation

CH3-(CH2)10

CH3-(CH2)10 CH3-(CH2)10

NH2

Kdo2-lipid A

R-3-OH-C14-ACP


O


l-Ara4N

O







AdaptorRapZ

AdaptorRapZ

AdaptorRapZ

AdaptorRapZ



SD

GlmS

GlcN6P UDP-GlcNAc


Peptidoglycan



Translation

Hfq

glmS mRNA

Complex

Processing

GlmZ

GlmY


glmS mRNA

RNase E

ProcessingglmS mRNA

SD Translation

Degradation

Complex

Processing

RNase E

UGA

GlmZ GlmZ

GlmY

GlmZ

GlmY

GlmZ GlmZ

GlmY

Free

Transcription

LowGlcN6P

HighGlcN6P

(a)

(b)

(c)

RapZ

RapZ




















2-lipid A) and











HO

HO

HO

HO

HO

OH

O

O

O

O

OO

OO

O

O

OH

HOO

O

O

O O

O

OH

O

OHO

OH

O

OO

OH

OH

OH

HO

P

P

NH

NH

12

1414

1414

HO

O

14

O

Lipid A

Kdo2

(a)

O

14

HO

HO

HO

OH

O

O

O

O

OO

O O

O

O

OH

HOO

O

OO O

O

OH

O

OO

OHO

O

OO

OOH

R2

OHHO

P

P

NH

NH

12

14

14 1414

HO

HOO

OH

O

R1 O

O

OHP

EptA

OO

OP P

LpxT

RdlA

MicA

OO

OH

EptBMgrR

LpxRMicF

ArcZ

O

16PagP

O

OHOHOH

NH2

NH2

PNH2

(b)









Lipid A

2

4

WaaAWaaQ7

1

PWaaP

44

PWaaY

5 KdoI 2

KdoII

WaaA

HepIII


WaaG

6

1WaaU

HepIV

GlcIII 1

WaaR

WaaB6

1

Gal

Glycoform I

WaaCGlcII 12

WaaO WaaF

(a)

Lipid A

2

4

PEptC

P-EtN

1

6

WaaSRha

2

4

WaaZ

Glycoform IV


Gal

HepI 1 53 KdoI 2

KdoII 5

1

7

44

P

MgrR

ArcZEptB

RybBWaaRRpoE

KdoIII

HepIII 1

RpoE

Hfq

MicAlowast

(b)

Lipid A

2

4

PEptC

P-EtN

1

6

EptB

2

4

WaaZ

Glycoform V


Gal

HepI 1 53 KdoI 2

KdoII 7

1

7

1

WaaSRha5

P-EtN

44

P

KdoIII

HepIII

RybBWaaRRpoE

RpoE

Hfq

RpoE

MicAlowast

(c)

Lipid A

2

4

PEptC

P-EtN

1

6

EptB

2

4

WaaZ


Gal HepIII

HepI 13 5 KdoI 2

KdoII 7

1

7

1

WaaSRha5

P-EtN

4

WaaH7

1

GlcUA

Glycoform VII

KdoIII

RybBWaaRRpoE

RpoE

HfqRpoE

MicAlowast

(d)


























11 Conclusion






Acknowledgments


References




































































































galK

Spot42

CRP

cAMP

Glucose uptake

SgrS

LPS

prec

urso

rs

Export of LPSto OM

Deacylation

Acylation with C16

LpxR

MicF

lpxR

pagP

PhoP

MicA

PagP


pmrR

BasSR

PmrR

LpxT

lpxT


O

OH

HOHO

OONH

UDP

LpxA

14

OOH

HO

O ONH UDP

O

HO

O

LpxC

FabZ O

ACP

FabIPhospholipids

PE

PG

UDP-GlcNAc

SlrA Lpp

Core biosynthesis

P-EtN onthe second

Kdo

eptBArcZ

MicAPhoPQ

P-EtN

GcvBMgrR

lpxT waaRGlcIII



GlmZY

waaZpmrD

BasSRglmS

waaB waaS

ACP

OH

ACP

14

OOH

HO

O UDP

O

HO

O

UDP-GlcUDP-Gal


arnTeptA


LPS translocation

CH3-(CH2)10

CH3-(CH2)10 CH3-(CH2)10

NH2

Kdo2-lipid A

R-3-OH-C14-ACP


O


l-Ara4N

O







AdaptorRapZ

AdaptorRapZ

AdaptorRapZ

AdaptorRapZ



SD

GlmS

GlcN6P UDP-GlcNAc


Peptidoglycan



Translation

Hfq

glmS mRNA

Complex

Processing

GlmZ

GlmY


glmS mRNA

RNase E

ProcessingglmS mRNA

SD Translation

Degradation

Complex

Processing

RNase E

UGA

GlmZ GlmZ

GlmY

GlmZ

GlmY

GlmZ GlmZ

GlmY

Free

Transcription

LowGlcN6P

HighGlcN6P

(a)

(b)

(c)

RapZ

RapZ




















2-lipid A) and











HO

HO

HO

HO

HO

OH

O

O

O

O

OO

OO

O

O

OH

HOO

O

O

O O

O

OH

O

OHO

OH

O

OO

OH

OH

OH

HO

P

P

NH

NH

12

1414

1414

HO

O

14

O

Lipid A

Kdo2

(a)

O

14

HO

HO

HO

OH

O

O

O

O

OO

O O

O

O

OH

HOO

O

OO O

O

OH

O

OO

OHO

O

OO

OOH

R2

OHHO

P

P

NH

NH

12

14

14 1414

HO

HOO

OH

O

R1 O

O

OHP

EptA

OO

OP P

LpxT

RdlA

MicA

OO

OH

EptBMgrR

LpxRMicF

ArcZ

O

16PagP

O

OHOHOH

NH2

NH2

PNH2

(b)









Lipid A

2

4

WaaAWaaQ7

1

PWaaP

44

PWaaY

5 KdoI 2

KdoII

WaaA

HepIII


WaaG

6

1WaaU

HepIV

GlcIII 1

WaaR

WaaB6

1

Gal

Glycoform I

WaaCGlcII 12

WaaO WaaF

(a)

Lipid A

2

4

PEptC

P-EtN

1

6

WaaSRha

2

4

WaaZ

Glycoform IV


Gal

HepI 1 53 KdoI 2

KdoII 5

1

7

44

P

MgrR

ArcZEptB

RybBWaaRRpoE

KdoIII

HepIII 1

RpoE

Hfq

MicAlowast

(b)

Lipid A

2

4

PEptC

P-EtN

1

6

EptB

2

4

WaaZ

Glycoform V


Gal

HepI 1 53 KdoI 2

KdoII 7

1

7

1

WaaSRha5

P-EtN

44

P

KdoIII

HepIII

RybBWaaRRpoE

RpoE

Hfq

RpoE

MicAlowast

(c)

Lipid A

2

4

PEptC

P-EtN

1

6

EptB

2

4

WaaZ


Gal HepIII

HepI 13 5 KdoI 2

KdoII 7

1

7

1

WaaSRha5

P-EtN

4

WaaH7

1

GlcUA

Glycoform VII

KdoIII

RybBWaaRRpoE

RpoE

HfqRpoE

MicAlowast

(d)


























11 Conclusion






Acknowledgments


References




































































































AdaptorRapZ

AdaptorRapZ

AdaptorRapZ

AdaptorRapZ



SD

GlmS

GlcN6P UDP-GlcNAc


Peptidoglycan



Translation

Hfq

glmS mRNA

Complex

Processing

GlmZ

GlmY


glmS mRNA

RNase E

ProcessingglmS mRNA

SD Translation

Degradation

Complex

Processing

RNase E

UGA

GlmZ GlmZ

GlmY

GlmZ

GlmY

GlmZ GlmZ

GlmY

Free

Transcription

LowGlcN6P

HighGlcN6P

(a)

(b)

(c)

RapZ

RapZ




















2-lipid A) and











HO

HO

HO

HO

HO

OH

O

O

O

O

OO

OO

O

O

OH

HOO

O

O

O O

O

OH

O

OHO

OH

O

OO

OH

OH

OH

HO

P

P

NH

NH

12

1414

1414

HO

O

14

O

Lipid A

Kdo2

(a)

O

14

HO

HO

HO

OH

O

O

O

O

OO

O O

O

O

OH

HOO

O

OO O

O

OH

O

OO

OHO

O

OO

OOH

R2

OHHO

P

P

NH

NH

12

14

14 1414

HO

HOO

OH

O

R1 O

O

OHP

EptA

OO

OP P

LpxT

RdlA

MicA

OO

OH

EptBMgrR

LpxRMicF

ArcZ

O

16PagP

O

OHOHOH

NH2

NH2

PNH2

(b)









Lipid A

2

4

WaaAWaaQ7

1

PWaaP

44

PWaaY

5 KdoI 2

KdoII

WaaA

HepIII


WaaG

6

1WaaU

HepIV

GlcIII 1

WaaR

WaaB6

1

Gal

Glycoform I

WaaCGlcII 12

WaaO WaaF

(a)

Lipid A

2

4

PEptC

P-EtN

1

6

WaaSRha

2

4

WaaZ

Glycoform IV


Gal

HepI 1 53 KdoI 2

KdoII 5

1

7

44

P

MgrR

ArcZEptB

RybBWaaRRpoE

KdoIII

HepIII 1

RpoE

Hfq

MicAlowast

(b)

Lipid A

2

4

PEptC

P-EtN

1

6

EptB

2

4

WaaZ

Glycoform V


Gal

HepI 1 53 KdoI 2

KdoII 7

1

7

1

WaaSRha5

P-EtN

44

P

KdoIII

HepIII

RybBWaaRRpoE

RpoE

Hfq

RpoE

MicAlowast

(c)

Lipid A

2

4

PEptC

P-EtN

1

6

EptB

2

4

WaaZ


Gal HepIII

HepI 13 5 KdoI 2

KdoII 7

1

7

1

WaaSRha5

P-EtN

4

WaaH7

1

GlcUA

Glycoform VII

KdoIII

RybBWaaRRpoE

RpoE

HfqRpoE

MicAlowast

(d)


























11 Conclusion






Acknowledgments


References


















































































































2-lipid A) and











HO

HO

HO

HO

HO

OH

O

O

O

O

OO

OO

O

O

OH

HOO

O

O

O O

O

OH

O

OHO

OH

O

OO

OH

OH

OH

HO

P

P

NH

NH

12

1414

1414

HO

O

14

O

Lipid A

Kdo2

(a)

O

14

HO

HO

HO

OH

O

O

O

O

OO

O O

O

O

OH

HOO

O

OO O

O

OH

O

OO

OHO

O

OO

OOH

R2

OHHO

P

P

NH

NH

12

14

14 1414

HO

HOO

OH

O

R1 O

O

OHP

EptA

OO

OP P

LpxT

RdlA

MicA

OO

OH

EptBMgrR

LpxRMicF

ArcZ

O

16PagP

O

OHOHOH

NH2

NH2

PNH2

(b)









Lipid A

2

4

WaaAWaaQ7

1

PWaaP

44

PWaaY

5 KdoI 2

KdoII

WaaA

HepIII


WaaG

6

1WaaU

HepIV

GlcIII 1

WaaR

WaaB6

1

Gal

Glycoform I

WaaCGlcII 12

WaaO WaaF

(a)

Lipid A

2

4

PEptC

P-EtN

1

6

WaaSRha

2

4

WaaZ

Glycoform IV


Gal

HepI 1 53 KdoI 2

KdoII 5

1

7

44

P

MgrR

ArcZEptB

RybBWaaRRpoE

KdoIII

HepIII 1

RpoE

Hfq

MicAlowast

(b)

Lipid A

2

4

PEptC

P-EtN

1

6

EptB

2

4

WaaZ

Glycoform V


Gal

HepI 1 53 KdoI 2

KdoII 7

1

7

1

WaaSRha5

P-EtN

44

P

KdoIII

HepIII

RybBWaaRRpoE

RpoE

Hfq

RpoE

MicAlowast

(c)

Lipid A

2

4

PEptC

P-EtN

1

6

EptB

2

4

WaaZ


Gal HepIII

HepI 13 5 KdoI 2

KdoII 7

1

7

1

WaaSRha5

P-EtN

4

WaaH7

1

GlcUA

Glycoform VII

KdoIII

RybBWaaRRpoE

RpoE

HfqRpoE

MicAlowast

(d)


























11 Conclusion






Acknowledgments


References




































































































HO

HO

HO

HO

HO

OH

O

O

O

O

OO

OO

O

O

OH

HOO

O

O

O O

O

OH

O

OHO

OH

O

OO

OH

OH

OH

HO

P

P

NH

NH

12

1414

1414

HO

O

14

O

Lipid A

Kdo2

(a)

O

14

HO

HO

HO

OH

O

O

O

O

OO

O O

O

O

OH

HOO

O

OO O

O

OH

O

OO

OHO

O

OO

OOH

R2

OHHO

P

P

NH

NH

12

14

14 1414

HO

HOO

OH

O

R1 O

O

OHP

EptA

OO

OP P

LpxT

RdlA

MicA

OO

OH

EptBMgrR

LpxRMicF

ArcZ

O

16PagP

O

OHOHOH

NH2

NH2

PNH2

(b)









Lipid A

2

4

WaaAWaaQ7

1

PWaaP

44

PWaaY

5 KdoI 2

KdoII

WaaA

HepIII


WaaG

6

1WaaU

HepIV

GlcIII 1

WaaR

WaaB6

1

Gal

Glycoform I

WaaCGlcII 12

WaaO WaaF

(a)

Lipid A

2

4

PEptC

P-EtN

1

6

WaaSRha

2

4

WaaZ

Glycoform IV


Gal

HepI 1 53 KdoI 2

KdoII 5

1

7

44

P

MgrR

ArcZEptB

RybBWaaRRpoE

KdoIII

HepIII 1

RpoE

Hfq

MicAlowast

(b)

Lipid A

2

4

PEptC

P-EtN

1

6

EptB

2

4

WaaZ

Glycoform V


Gal

HepI 1 53 KdoI 2

KdoII 7

1

7

1

WaaSRha5

P-EtN

44

P

KdoIII

HepIII

RybBWaaRRpoE

RpoE

Hfq

RpoE

MicAlowast

(c)

Lipid A

2

4

PEptC

P-EtN

1

6

EptB

2

4

WaaZ


Gal HepIII

HepI 13 5 KdoI 2

KdoII 7

1

7

1

WaaSRha5

P-EtN

4

WaaH7

1

GlcUA

Glycoform VII

KdoIII

RybBWaaRRpoE

RpoE

HfqRpoE

MicAlowast

(d)


























11 Conclusion






Acknowledgments


References




































































































Lipid A

2

4

WaaAWaaQ7

1

PWaaP

44

PWaaY

5 KdoI 2

KdoII

WaaA

HepIII


WaaG

6

1WaaU

HepIV

GlcIII 1

WaaR

WaaB6

1

Gal

Glycoform I

WaaCGlcII 12

WaaO WaaF

(a)

Lipid A

2

4

PEptC

P-EtN

1

6

WaaSRha

2

4

WaaZ

Glycoform IV


Gal

HepI 1 53 KdoI 2

KdoII 5

1

7

44

P

MgrR

ArcZEptB

RybBWaaRRpoE

KdoIII

HepIII 1

RpoE

Hfq

MicAlowast

(b)

Lipid A

2

4

PEptC

P-EtN

1

6

EptB

2

4

WaaZ

Glycoform V


Gal

HepI 1 53 KdoI 2

KdoII 7

1

7

1

WaaSRha5

P-EtN

44

P

KdoIII

HepIII

RybBWaaRRpoE

RpoE

Hfq

RpoE

MicAlowast

(c)

Lipid A

2

4

PEptC

P-EtN

1

6

EptB

2

4

WaaZ


Gal HepIII

HepI 13 5 KdoI 2

KdoII 7

1

7

1

WaaSRha5

P-EtN

4

WaaH7

1

GlcUA

Glycoform VII

KdoIII

RybBWaaRRpoE

RpoE

HfqRpoE

MicAlowast

(d)


























11 Conclusion






Acknowledgments


References

























































































































11 Conclusion






Acknowledgments


References







































































































Acknowledgments


References



































































































Regulated Control of the Assembly and Diversity of LPS by ... · Ugd BasS/R PmrR LpxT EptC Rcs RprA...

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